Anti-connexin antibody formulations

ABSTRACT

The present disclosure relates to pharmaceutical compositions and methods for treating a disease or condition associated with opening of Cx43 hemichannels in astrocytes or osteocytes, preferably for treating an inflammatory disease or condition or a neurodegenerative disease such as spinal cord injury.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 63/059,502 filed Jul. 31, 2020, the entiredisclosure of which is incorporated herein by reference.

SEQUENCE LISTING

The ASCII text file submitted herewith via EFS-Web, entitled “020601SequenceListing.txt” created on Jul. 30, 2021, having a size of 41,779bytes, is hereby incorporated by reference in its entirety.

FIELD

The present disclosure generally relates to stable aqueouspharmaceutical compositions comprising anti-connexin (Cx) 43 antibodies.

BACKGROUND

Antibodies (Abs) have been used in the treatment of various diseases andconditions due to their specificity of target recognition, therebygenerating highly selective outcomes following systemic administration.In order for antibodies to remain effective, they must maintain theirbiological activity during their production, purification, transport andstorage. New production and purification techniques have been developedto provide for large amounts of highly purified monoclonal antibodies tobe produced. However, challenges still exist to stabilize theseantibodies for transport and storage, and yet even more challenges existto provide the antibodies in a dosage form suitable for administration.

Denaturation, aggregation, contamination, and particle formation can besignificant obstacles in the formulation and storage of antibodies. Dueto the wide variety of antibodies, there are no universal formulationsor conditions suitable for storage of all antibodies. Optimalformulations and conditions suitable for storage of one antibody areoften specific to that antibody. Thus, antibody storage formulations andmethods are often a significant part of the research and developmentprocess for a commercial antibody.

Various methods have been proposed to overcome the challenges associatedwith antibody stability. For example, in some instances, the antibody isoften lyophilized, and then reconstituted shortly before administration.However, reconstitution is generally not ideal, since it adds anadditional step to the administration process, and could introducecontaminants to the formulation. Additionally, even reconstitutedantibodies can suffer from aggregation and particle formation. Thus, aneed exists to provide stable, aqueous antibody formulations, inparticular anti-Cx43 antibody formulations that can overcome thechallenges associated with transport and storage.

SUMMARY

The present disclosure provides, in one aspect, a pharmaceuticalformulation comprising:

-   -   an anti-Cx43 antibody (Ab) or antigen binding fragment thereof;    -   a buffer;    -   a surfactant; and    -   a stabilizer;    -   wherein the pharmaceutical formulation has a pH of between about        5 and about 6;    -   wherein the anti-Cx43 antibody or antigen binding fragment        thereof comprises:        -   a first, second and third heavy chain complementarity            determining region (CDR) sequence having the amino acid            sequence of SEQ ID NOs: 1, 2, and 3, respectively; and        -   a first, second and third light chain CDR sequence having            the amino acid sequence of SEQ ID NOs: 4, 5, and 6,            respectively.

In some embodiments, the anti-Cx43 antibody or antigen binding fragmentthereof comprises a heavy chain variable domain having the amino acidsequence of SEQ ID NO: 7, and a light chain variable domain having theamino acid sequence of SEQ ID NO: 8.

In certain embodiments, the anti-Cx43 antibody or antigen bindingfragment thereof comprises a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9-17, and a lightchain having the amino acid sequence of SEQ ID NO: 18.

In certain embodiments, the anti-Cx43 antibody or antigen bindingfragment thereof binds to an epitope located within the amino acidsequence of FLSRPTEKTI (SEQ ID NO: 19). In some embodiments, the epitopecomprises one or more amino acids selected from the group consisting ofR4, P5, E7, K8 and I10 of SEQ ID NO: 19. In some embodiments, theepitope consists of R4, P5, E7, K8 and I10 of SEQ ID NO: 19. In someembodiments, the epitope comprises all ten amino acids of SEQ ID NO: 19.In some embodiments, the epitope consists of all ten amino acids of SEQID NO: 19.

In some embodiments, the anti-Cx43 antibody or antigen binding fragmentthereof is present at a concentration of between about 5 and about 100mg/mL, preferably between 20 and 80, more preferably from about 40 to 60mg/mL.

In some certain embodiments, the buffer is selected from acetate/sodiumacetate, histidine/aspartic acid, citric acid/sodium citrate, dibasicsodium phosphate/sodium dihydrogen phosphate, and histidine/histidinehydrochloride. In certain embodiments, the buffer is histidine/asparticacid or histidine/histidine hydrochloride. In certain embodiments, thebuffer is histidine/histidine hydrochloride.

In some embodiments, the surfactant is polysorbate 80 (PS80).

In certain embodiments, the stabilizer is selected fromethylenediaminetetraacetic acid (EDTA), sodium chloride, sorbitol,glycine, and sucrose. In certain embodiments, the stabilizer is sucrose.

In certain embodiments, the pH of the formulation is between about 5.4to about 5.6.

In some embodiments, the formulation is an aqueous formulation. In someembodiments, the formulation is a stable aqueous formulation.

Another aspect relates to a pharmaceutical formulation comprising:

-   -   about 40-60 mg/mL, preferably about 50 mg/mL of an anti-Cx43        antibody or antigen binding fragment thereof;    -   about 10-40 mM, preferably about 20 mM histidine/histidine        hydrochloride buffer;    -   about 0.005%-0.05%, preferably about 0.02% w/v Polysorbate 80;        and    -   about 1%-20% w/v, preferably about 8% w/v sucrose;    -   wherein the formulation has a pH of between about 5.4 to about        5.6, preferably about 5.5.

A further aspect relates to a pharmaceutical formulation comprising:

-   -   about 50 mg/mL an anti-Cx43 antibody or antigen binding fragment        thereof, comprising a heavy chain having an amino acid sequence        selected from the group consisting of SEQ ID NOs: 9-17, and        comprising a light chain having the amino acid sequence of SEQ        ID NO: 18;    -   about 20 mM histidine/aspartic acid buffer;    -   about 0.02% w/v Polysorbate 80; and    -   about 8% w/v sucrose,    -   wherein the formulation has a pH of between about 5.4 to about        5.6, preferably about 5.5.

Kits and/or unit dosages comprising any one of the pharmaceuticalformulations disclosed herein are also provided.

Also provided herein is use of any one of the pharmaceuticalformulations disclosed herein, for inhibiting opening of Cx43hemichannels in astrocytes or osteocytes, preferably for treating aninflammatory disease or condition or a neurodegenerative disease such asspinal cord injury.

Additionally provided herein is a method of inhibiting opening of Cx43hemichannels in cells, comprising administering to a subject in needthereof any one of the pharmaceutical formulations disclosed herein. Insome embodiments, the method can be used for treating an inflammatorydisease or condition or a neurodegenerative disease such as spinal cordinjury.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: MicroCal DSC thermogram overlay from the anti-Cx43 Ab pH/Bufferscreening study.

FIG. 2: SEC-Main peak % comparison at 25±2° C. (left) and 40±2° C.(right) from the pH/buffer screening study.

FIG. 3: Comparison of cIEF main peak % at 25±2° C. (left) and 40±2° C.(right) from the pH/buffer screening study.

FIG. 4: Non-reduced SDS-Caliper purity % comparison from the pH/Bufferscreening study at 25±2° C. (left) and 40±2° C. (right).

FIG. 5: Reduced SDS-Caliper purity % comparison from the pH/Bufferscreening study at 25±2° C. (left) and 40±2° C. (right).

FIG. 6: SEC-HPLC main peak % comparison from the freeze/thaw study.

FIG. 7: Comparison of cIEF main peak % from the freeze/thaw study.

FIG. 8: Purity % comparison from the freeze/thaw study in non-reducedSDS-Caliper (left) and reduced SDS-Caliper (right).

FIG. 9: SEC-HPLC main peak % comparison from the agitation study.

FIG. 10: Comparison of cIEF main peak % from the agitation study.

FIG. 11: Purity % comparison from the agitation study in non-reducedSDS-Caliper (left) and reduced SDS-Caliper (right).

FIG. 12: SEC-Main peak % comparison at 2˜8° C. (left), 25±2° C. (middle)and 40±2° C. (right).

FIG. 13: cIEF main peak % comparison at 2˜8° C. (left), 25±2° C.(middle) and 40±2° C. (right).

FIG. 14: Non-reduced SDS-Caliper purity % comparison at 2˜8° C. (left),25±2° C. (middle) and 40±2° C. (right).

FIG. 15: Reduced SDS-Caliper purity % comparison at 2˜8° C. (left),25±2° C. (middle) and 40±2° C. (right).

FIG. 16: MicroCal DSC thermogram overlay from the anti-Cx43 Abformulation confirmation study.

DETAILED DESCRIPTION

Disclosed herein, in some embodiments, is a stable, aqueouspharmaceutical formulation of anti-Cx43 antibodies. Such formulation caninclude: an anti-Cx43 antibody or antigen binding fragment thereof, abuffer, a surfactant, and a stabilizer. The pharmaceutical formulationcan have a pH of between about 5 and about 6, or about 5.4-5.6, or about5.5.

In some embodiments, the anti-Cx43 antibody or antigen binding fragmentthereof can have a first, second and third heavy chain complementaritydetermining region (CDR) sequence having the amino acid sequence of SEQID NOs: 1, 2, and 3, respectively; and/or a first, second and thirdlight chain CDR sequence having the amino acid sequence of SEQ ID NOs:4, 5, and 6, respectively.

In some embodiments, the anti-Cx43 antibody or antigen binding fragmentthereof can have a heavy chain variable domain having the amino acidsequence of SEQ ID NO: 7, and a light chain variable domain having theamino acid sequence of SEQ ID NO: 8.

In certain embodiments, the anti-Cx43 antibody or antigen bindingfragment thereof comprises a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9-17, and a lightchain having the amino acid sequence of SEQ ID NO: 18.

In certain embodiments, the anti-Cx43 antibody or antigen bindingfragment thereof binds to an epitope located within the amino acidsequence of FLSRPTEKTI (SEQ ID NO: 19).

In various embodiments, the formulations disclosed herein can haveimproved stability, such that they display no significant changes (suchas appearance, antibody concentration, pH, antibody aggregation, andantibody purity) observed at a predetermined temperature (e.g., −40° C.or −20° C. or refrigerated temperature of 2-8° C.) for a period of time,e.g., at least 3 months, at least 6 months, at least 1 year, or up to 3years.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this disclosure pertains. The following referencesprovide one of skill with a general definition of many of the terms usedin this disclosure: Academic Press Dictionary of Science and Technology,Morris (Ed.), Academic Press (1^(st) ed., 1992); Oxford Dictionary ofBiochemistry and Molecular Biology, Smith et al. (Eds.), OxfordUniversity Press (revised ed., 2000); Encyclopaedic Dictionary ofChemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionaryof Microbiology and Molecular Biology, Singleton et al. (Eds.), JohnWiley & Sons (3^(rd) ed., 2002); Dictionary of Chemistry, Hunt (Ed.),Routledge (1^(st) ed., 1999); Dictionary of Pharmaceutical Medicine,Nahler (Ed.), Springer-Verlag Telos (1994); Dictionary of OrganicChemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd.(2002); and A Dictionary of Biology (Oxford Paperback Reference), Martinand Hine (Eds.), Oxford University Press (4^(th) ed., 2000). Furtherclarifications of some of these terms as they apply specifically to thisdisclosure are provided herein.

As used herein, the articles “a” and “an” refer to one or more than one,e.g., to at least one, of the grammatical object of the article. The useof the words “a” or “an” when used in conjunction with the term“comprising” herein may mean “one,” but it is also consistent with themeaning of “one or more,” “at least one,” and “one or more than one.”

As used herein, “about” and “approximately” generally mean an acceptabledegree of error for the quantity measured given the nature or precisionof the measurements. Exemplary degrees of error are within 20 percent(%), typically, within 10%, and more typically, within 5% of a givenrange of values. The term “substantially” means more than 50%,preferably more than 80%, and most preferably more than 90% or 95%.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that arepresent in a given embodiment, yet open to the inclusion of unspecifiedelements.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof additional elements that do not materially affect the basic and novelor functional characteristic(s) of that embodiment of the disclosure.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

An “anti-Cx43 antibody” is an antibody that immunospecifically binds toCx43 (e.g., its extracellular domain). The antibody may be an isolatedantibody. Such binding to Cx43 exhibits a KD with a value of, e.g., nogreater than 1 μM, no greater than 100 nM or no greater than 50 nM. KDcan be measured by any methods known to one skilled in the art, such asa surface plasmon resonance assay or a cell binding assay. An anti-Cx43antibody may be a monoclonal antibody, or antigen-binding fragmentsthereof. In some embodiments, the antibody can be those disclosed in PCTApplication No. PCT/US2020/016606 filed Feb. 4, 2020, incorporatedherein by reference in its entirety.

An “antibody,” as used herein is a protein comprising binding domainsthat bind to a target epitope. The term antibody includes monoclonalantibodies comprising immunoglobulin heavy and light chain molecules,single heavy chain variable domain antibodies, and variants andderivatives thereof, including chimeric variants of monoclonal andsingle heavy chain variable domain antibodies. Binding domains aresubstantially encoded by immunoglobulin genes or fragments ofimmunoglobulin genes, wherein the protein immunospecifically binds to anantigen. The recognized immunoglobulin genes include the kappa, lambda,alpha, gamma, delta, epsilon and mu constant region genes, as well asmyriad immunoglobulin variable region genes. Light chains are classifiedas either kappa or lambda. Heavy chains are classified as gamma, mu,alpha, delta, or epsilon, which in turn define the immunoglobulinclasses, IgG, IgM, IgA, IgD and IgE, respectively. For most vertebrateorganisms, including humans and murine species, the typicalimmunoglobulin structural unit comprises a tetramer that is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kD) and one “heavy” chain (about 50-70 kD). “V_(L)” and V_(H)”refer to the variable domains of these light and heavy chainsrespectively. “C_(L)” and C_(H)” refer to the constant domains of thelight and heavy chains. Loops of β-strands, three each on the V_(L) andV_(H) are responsible for binding to the antigen, and are referred to asthe “complementarity determining regions” or “CDRs”. The “Fab”(fragment, antigen-binding) region includes one constant and onevariable domain from each heavy and light chain of the antibody, i.e.,V_(L), C_(L), V_(H) and C_(H).

Antibodies include intact immunoglobulins as well as antigen-bindingfragments thereof The term “antigen-binding fragment” refers to apolypeptide fragment of an antibody which binds antigen or competes withintact antibody (i.e., with the intact antibody from which they werederived) for antigen binding (i.e., specific binding). Antigen bindingfragments can be produced by recombinant or biochemical methods that arewell known in the art. Exemplary antigen-binding fragments include Fv,Fab, Fab′, (Fab′)₂, CDR, paratope and single chain Fv antibodies (scFv)in which a V_(H) and a V_(L) chain are joined together (directly orthrough a peptide linker) to form a continuous polypeptide.

Antibodies also include variants, chimeric antibodies and humanizedantibodies. The term “antibody variant” as used herein refers to anantibody with single or multiple mutations in the heavy chains and/orlight chains. In some embodiments, the mutations exist in the variableregion. In some embodiments, the mutations exist in the constant region.“Chimeric antibodies” refers to those antibodies wherein one portion ofeach of the amino acid sequences of heavy and light chains is homologousto corresponding sequences in antibodies derived from a particularspecies or belonging to a particular class, while the remaining segmentof the chains is homologous to corresponding sequences in another.Typically, in these chimeric antibodies, the variable region of bothlight and heavy chains mimics the variable regions of antibodies derivedfrom one species of mammals, while the constant portions are homologousto the sequences in antibodies derived from another. One clear advantageto such chimeric forms is that, for example, the variable regions canconveniently be derived from presently known sources using readilyavailable hybridomas or B cells from non-human host organisms incombination with constant regions derived from, for example, human cellpreparations. While the variable region has the advantage of ease ofpreparation, and the specificity is not affected by its source, theconstant region being human, is less likely to elicit an immune responsefrom a human subject when the antibodies are injected than would theconstant region from a non-human source. However, the definition is notlimited to this particular example. “Humanized” antibodies refer to amolecule having an antigen-binding site that is substantially derivedfrom an immunoglobulin from a non-human species and the remainingimmunoglobulin structure of the molecule based upon the structure and/orsequence of a human immunoglobulin. The antigen-binding site maycomprise either complete variable domains fused onto constant domains oronly the complementarity determining regions (CDRs) grafted ontoappropriate framework regions in the variable domains. Antigen bindingsites may be wild type or modified by one or more amino acidsubstitutions, e.g., modified to resemble human immunoglobulin moreclosely. Some forms of humanized antibodies preserve all CDR sequences(for example, a humanized mouse antibody which contains all six CDRsfrom the mouse antibodies). Other forms of humanized antibodies have oneor more CDRs (one, two, three, four, five, or six) which are alteredwith respect to the original antibody, which are also termed one or moreCDRs “derived from” one or more CDRs.

As described herein, the amino acid residues of an antibody can benumbered according to the general numbering of Kabat (Kabat, et al.(1991) Sequences of Proteins of Immunological Interest, 5th edition.Public Health Service, NIH, Bethesda, Md.).

The term “binding” as used herein in the context of binding between anantibody and an epitope of Cx43 as a target, refers to the process of anon-covalent interaction between molecules. Preferably, said binding isspecific. The specificity of an antibody can be determined based onaffinity. A specific antibody can have a binding affinity ordissociation constant KD for its epitope of less than 10⁻⁷ M, preferablyless than 10⁻⁸ M.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as anantibody, and additionally capable of being used in an animal to produceantibodies capable of binding to an epitope of that antigen. An antigenmay have one or more epitopes.

The term “epitope” includes any determinant, preferably a polypeptidedeterminant, capable of specific binding to an immunoglobulin or T-cellreceptor. In certain embodiments, epitope determinants includechemically active surface groupings of molecules such as amino acids,sugar side chains, phosphoryl, or sulfonyl, and, in certain embodiments,may have specific three-dimensional structural characteristics, and/orspecific charge characteristics. In one embodiment, an epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. Methods for epitope mapping are wellknown in the art, such as X-ray co-crystallography, array-basedoligo-peptide scanning, site-directed mutagenesis, high throughputmutagenesis mapping and hydrogen-deuterium exchange. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents, whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5 or 8-10 amino acids in a uniquespatial conformation.

The term “subject” or “patient” includes a human or other mammaliananimal that receives either prophylactic or therapeutic treatment.

The terms “treat,” “treating,” and “treatment,” as used herein, refer totherapeutic or preventative measures such as those described herein. Themethods of “treatment” employ administration to a patient a Cx43 ligandprovided herein, for example, a patient having an inflammatory diseaseor condition or a neurodegenerative disease, in order to prevent, cure,delay, reduce the severity of, or ameliorate one or more symptoms of theinflammatory disease or condition or a neurodegenerative disease, or inorder to prolong the survival of a patient beyond that expected in theabsence of such treatment. The methods of “treatment” also employadministration to a patient a Cx43 ligand provided herein (e.g., anantibody) to provide therapy in a patient beyond that expected in theabsence of such treatment.

The term “inflammatory disease” broadly refers to the vast array ofdisorders and conditions that are characterized by inflammation.Examples include arthritis, allergy, asthma, autoimmune diseases,coeliac disease, glomerulonephritis, hepatitis, inflammatory boweldisease (including Crohn's disease and Ulcerative Colitis), reperfusioninjury and transplant rejection.

The term “autoimmune disease” broadly refers to diseases in which theimmune system attacks its own proteins, cells, and tissues, or in whichimmune effector T cells are autoreactive to endogenous self peptides andcause destruction of tissue. Autoimmune diseases include but are notlimited to rheumatoid arthritis, Crohn's disease, Type 1 diabetes,alopecia, multiple sclerosis, lupus, systemic lupus erythematosus (SLE),autoimmune encephalomyelitis, myasthenia gravis (MG), Hashimoto'sthyroiditis, Goodpasture's syndrome, pemphigus (e.g., pemphigusvulgaris), Grave's disease, autoimmune hemolytic anemia, autoimmunethrombocytopenic purpura, scleroderma with anti-collagen antibodies,mixed connective tissue disease, polymyositis, pernicious anemia,idiopathic Addison's disease, autoimmune-associated infertility,glomerulonephritis (e.g., crescentic glomerulonephritis, proliferativeglomerulonephritis), bullous pemphigoid, Sjogren's syndrome, insulinresistance, and autoimmune diabetes mellitus.

The term “neurodegenerative disease” broadly refers to diseasescharacterized by the progressive loss of structure and/or function ofneurons. Neurodegenerative diseases include but are not limited toAlzheimer's disease (AD), lysosomal storage disorders, bacterialmeningitis, amyotrophic lateral sclerosis, hypoxia, ischemia, glaucoma,schizophrenia, major depression, bipolar disorder, epilepsy, traumaticbrain injury, post-traumatic stress disorder, Parkinson's disease, Downsyndrome, spinocerebellar ataxia, Huntington's disease, radiationtherapy induced neurodegeneration, chronic stress inducedneurodegeneration, and neurodegeneration associated with normal aging orabuse of neuro-active drugs (such as alcohol, opiates, methamphetamine,phencyclidine, and cocaine).

The term “effective amount” as used herein, refers to that amount of anagent, such as a Cx43 ligand, for example an anti-Cx43 antibody, whichis sufficient to effect treatment, prognosis or diagnosis of a disease,when administered to a patient. A therapeutically effective amount willvary depending upon the patient and disease condition being treated, theweight and age of the patient, the severity of the disease condition,the manner of administration and the like, which can readily bedetermined by one of ordinary skill in the art. The dosages foradministration can range from, for example, about 1 ng to about 10,000mg, about 5 ng to about 9,500 mg, about 10 ng to about 9,000 mg, about20 ng to about 8,500 mg, about 30 ng to about 7,500 mg, about 40 ng toabout 7,000 mg, about 50 ng to about 6,500 mg, about 100 ng to about6,000 mg, about 200 ng to about 5,500 mg, about 300 ng to about 5,000mg, about 400 ng to about 4,500 mg, about 500 ng to about 4,000 mg,about 1 μg to about 3,500 mg, about 5 μg to about 3,000 mg, about 10 μgto about 2,600 mg, about 20 μg to about 2,575 mg, about 30 μg to about2,550 mg, about 40 μg to about 2,500 mg, about 50 μg to about 2,475 mg,about 100 μg to about 2,450 mg, about 200 μg to about 2,425 mg, about300 μg to about 2,000, about 400 μg to about 1,175 mg, about 500 μg toabout 1,150 mg, about 0.5 mg to about 1,125 mg, about 1 mg to about1,100 mg, about 1.25 mg to about 1,075 mg, about 1.5 mg to about 1,050mg, about 2.0 mg to about 1,025 mg, about 2.5 mg to about 1,000 mg,about 3.0 mg to about 975 mg, about 3.5 mg to about 950 mg, about 4.0 mgto about 925 mg, about 4.5 mg to about 900 mg, about 5 mg to about 875mg, about 10 mg to about 850 mg, about 20 mg to about 825 mg, about 30mg to about 800 mg, about 40 mg to about 775 mg, about 50 mg to about750 mg, about 100 mg to about 725 mg, about 200 mg to about 700 mg,about 300 mg to about 675 mg, about 400 mg to about 650 mg, about 500mg, or about 525 mg to about 625 mg, of an antibody or antigen bindingportion thereof, as provided herein. Dosing may be, e.g., every week,every 2 weeks, every three weeks, every 4 weeks, every 5 weeks or every6 weeks. Dosage regimens may be adjusted to provide the optimumtherapeutic response. An effective amount is also one in which any toxicor detrimental effects (side effects) of the agent are minimized and/oroutweighed by the beneficial effects. Administration may be intravenousat exactly or about 6 mg/kg or 12 mg/kg weekly, or 12 mg/kg or 24 mg/kgbiweekly. Additional dosing regimens are described below.

As used herein, “formulation” is a composition of a pharmaceuticallyactive drug, such as a biologically active protein (e.g., antibody),that is suitable for parenteral administration (including but notlimited to intravenous, intramuscular, or subcutaneous) to a patient inneed thereof and includes only pharmaceutically acceptable excipients,diluents, and other additives deemed safe by the Federal DrugAdministration or other foreign national authorities.

As used herein the phrases “liquid formulation” and “aqueousformulation” are used interchangeably to refer to a solution or liquidpreparation that contains a biopharmaceutical in combination with one ormore excipients (e.g., chemical additives)—dissolved in a suitablesolvent.

A “stable” formulation is a pharmaceutical formulation with nosignificant changes observed at a predetermined temperature (e.g., −40°C. or −20° C. or refrigerated temperature of 2-8° C.) for a period oftime, e.g., at least 3 months, at least 6 months, at least 1 year, or upto 3 years. Stability of the formulations disclosed herein can beevaluated using one or more of the following criteria: 1) the aqueousformulation is colorless, or clear to slightly opalescent by visualanalysis; 2) the protein content is maintained within +/−5 mg/mL frominitial concentration; 3) the pH is maintained within +/−0.5 pH unitsfrom target pH; 4) the percent of monomer by SEC is ≥95%; 5) the purityas measured by CE-SDS is ≥90% and the relative potency based on ELISA iswithin 60-150%.

As used herein the term “excipient” is intended to mean atherapeutically inactive substance. Excipients are included in aformulation for a wide variety of purposes, for example, as a buffer,stabilizer, tonicity agent, surfactant, anti-oxidant, cryoprotectant ordiluent.

Suitable excipients include, but are not limited to polyols (also knownas sugar alcohols) such as mannitol or sorbitol, sugars such as sucrose,lactose or dextrose, salts such as NaCl, KCl or calcium phosphate, aminoacids, for example, histidine, lysine, aspartic acid, or glutamic acid,surfactants, as well as water. The purity of the excipient should meetcompendial standards (e.g., USP, EP, JP) and be of sufficient purity forsubcutaneous, intramuscular, or intravenous injection into humans.

The term “buffer” or “buffering agent” as used herein, refers to apharmaceutically acceptable excipient, which stabilizes the pH of apharmaceutical preparation. Suitable buffers are well known in the artand can be found in the literature. For example, citrate salts, acetatesalts, histidine salts, succinate salts, malate salts, phosphate saltsor lactate salts, and/or the respective free acids or bases thereof, aswell as mixtures of the various salts and/or acids and bases thereof canbe employed. In a particular embodiment, pharmaceutically acceptablebuffers comprise but are not limited to histidine buffers, citratebuffers, succinate buffers, acetate buffers and phosphate buffers. In aparticular embodiment, buffers are acetate buffers, for example, sodiumacetate buffer. Other particular buffers are histidine buffers, i.e.buffers having histidine, generally L-histidine, as buffering agent. Aparticular buffer is L-histidine/HC1 buffer, comprising L-histidine ormixtures of L-histidine and L-histidine hydrochloride and pH adjustmentachieved with hydrochloric acid. Unless otherwise indicated, the term“L-histidine” when used herein to describe a buffering agent, refers toL-histidine/HCl buffer. L-histidine/HCl buffer can be prepared bydissolving suitable amounts of L-histidine and L-histidine hydrochloridein water, or by dissolving a suitable amount of L-histidine in water andadjusting the pH to the desired value by addition of hydrochloric acid.The abovementioned buffers are generally used at a concentration ofabout 1 mM to about 100 mM, about 10 mM to about 50 mM, about 15 to 30mM or 20 mM. Regardless of the buffer used, the pH can be adjusted to avalue in the range from about 4.0 to about 7.0, about 5.0 to about 6.0,about 5.4 to about 5.6, or about 5.5, with an acid or a base known inthe art, e.g., hydrochloric acid, acetic acid, phosphoric acid, sulfuricacid and citric acid, sodium hydroxide and potassium hydroxide.

The term “surfactant” as used herein denotes a pharmaceuticallyacceptable, surface-active agent. In a particular embodiment, anon-ionic surfactant is used. Examples of pharmaceutically acceptablesurfactants include, but are not limited to, polyoxyethylen-sorbitanfatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij),alkylphenylpolyoxyethylene ethers (Triton X),polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), andsodium dodecyl sulphate (SDS). In a particular embodiment,polyoxyethylene-sorbitan fatty acid esters are polysorbate 20(polyoxyethylene sorbitan monolaureate, sold under the trademark Tween20™) and polysorbate 80 (polyoxyethylene sorbitan monooleate, sold underthe trademark Tween 80™). In a particular embodiment,polyethylene-polypropylene copolymers are those sold under the namesPluronic® F68 or Poloxamer 188™. In a particular embodiment,polyoxyethylene alkyl ethers are those sold under the trademark Brij™.In a particular embodiment, alkylphenylpolyoxyethylene ethers are soldunder the tradename Triton X, for example, p-tert-octylphenoxypolyethoxyethanol (sold under the tradename Triton X-100™). Whenpolysorbate 20 (Tween 20™) and polysorbate 80 (Tween 80™) are used, theyare generally used at a concentration range of about 0.001 to about 1%,about 0.01 to about 0.1% or about 0.02% to about 0.05%. In theformulation of the disclosure, the concentration of the surfactant isdescribed as a percentage, expressed in weight/volume (w/v).

The term “stabilizer” as used herein denotes a pharmaceuticallyacceptable excipient, which protects the active pharmaceuticalingredient and/or the formulation from chemical and/or physicaldegradation during manufacturing, storage and application. Stabilizersinclude but are not limited to saccharides, amino acids, polyols, e.g.mannitol, sorbitol, xylitol, dextran, glycerol, arabitol, propyleneglycol, polyethylene glycol, cyclodextrines, e.g.hydroxypropyl-β-cyclodextrine, sulfobutylethyl-β-cyclodextrine,β-cyclodextrine, polyethylenglycols, e.g. PEG 3000, PEG 3350, PEG 4000,PEG 6000, albumines, e.g. human serum albumin (HSA), bovine serumalbumin (BSA), salts, e.g. sodium chloride, magnesium chloride, calciumchloride, chelators, e.g. EDTA as hereafter defined. As mentionedhereinabove, stabilizers can be present in the formulation in an amountof about 1 to about 500 mM, in an amount of about 10 to about 300 mM orin an amount of about 120 mM to about 300 mM. More than one stabilizer,selected from the same or from different groups, can be present in theformulation.

The term “saccharide” as used herein includes monosaccharides andoligosaccharides. A monosaccharide is a monomeric carbohydrate which isnot hydrolysable by acids, including simple sugars and theirderivatives, e.g. aminosugars. Saccharides are usually in their Dconformation. Examples of monosaccharides include glucose, fructose,galactose, mannose, sorbose, ribose, deoxyribose, neuraminic acid. Anoligosaccharide is a carbohydrate consisting of more than one monomericsaccharide unit connected via glycosidic bond(s) either branched or in alinear chain. The monomeric saccharide units within an oligosaccharidecan be identical or different. Depending on the number of monomericsaccharide units the oligosaccharide is a di-, tri-, tetra- penta- andso forth saccharide. In contrast to polysaccharides the monosaccharidesand oligosaccharides are water soluble. Examples of oligosaccharidesinclude sucrose, trehalose, lactose, maltose and raffinose. In aparticular embodiment, saccharides are sucrose and trehalose (i.e.α,α-D-trehalose), for example, sucrose. Trehalose is available astrehalose dihydrate. Saccharides can be present in the formulation in anamount of about 100 to about 500 mM, in an amount of about 200 to about300 mM or in an amount of about 240 mM.

A subgroup within the stabilizers are lyoprotectants. The term“lyoprotectant” denotes pharmaceutically acceptable excipients, whichprotect the labile active ingredient (e.g. a protein) againstdestabilizing conditions during the lyophilisation process, subsequentstorage and reconstitution. Lyoprotectants comprise but are not limitedto the group consisting of saccharides, polyols (such as e.g. sugaralcohols) and amino acids. In a particular embodiment, lyoprotectantscan be selected from the group consisting of saccharides such assucrose, trehalose, lactose, glucose, mannose, maltose, galactose,fructose, sorbose, raffinose, neuraminic acid, amino sugars such asglucosamine, galactosamine, N-methylglucosamine (“Meglumine”), polyolssuch as mannitol and sorbitol, and amino acids such as arginine andglycine or mixtures thereof. Lyoprotectants are generally used in anamount of about 10 to 500 mM, in an amount of about 10 to about 300 mMor in an amount of about 100 to about 300 mM.

Another subgroup within the stabilizers are antioxidants. The term“antioxidant” denotes pharmaceutically acceptable excipients, whichprevent oxidation of the active pharmaceutical ingredient. Antioxidantscomprise but are not limited to ascorbic acid, gluthathione, cysteine,methionine, citric acid, EDTA. Antioxidants can be used in an amount ofabout 0.01 to about 100 mM, in an amount of about 5 to about 50 mM or inan amount of about 5 to about 25 mM.

The formulations according to the disclosure may also comprise one ormore tonicity agents. The term “tonicity agents” denotespharmaceutically acceptable excipients used to modulate the tonicity ofthe formulation. The formulation can be hypotonic, isotonic orhypertonic. Isotonicity in general relates to the osmotic pressure of asolution, usually relative to that of human blood serum (around 250-350mOsmol/kg). The formulation according to the disclosure can behypotonic, isotonic or hypertonic. In a particular embodiment, theformulation is isotonic. An isotonic formulation is liquid or liquidreconstituted from a solid form, e.g. from a lyophilized form, anddenotes a solution having the same tonicity as some other solution withwhich it is compared, such as physiologic salt solution and the bloodserum. Suitable tonicity agents comprise but are not limited to sodiumchloride, potassium chloride, glycerine and any component from the groupof amino acids or sugars, in particular glucose. Tonicity agents aregenerally used in an amount of about 5 mM to about 500 mM.

Within the stabilizers and tonicity agents there is a group of compoundswhich can function in both ways, i.e. they can at the same time be astabilizer and a tonicity agent. Examples thereof can be found in thegroup of sugars, amino acids, polyols, cyclodextrines,polyethyleneglycols and salts. An example for a sugar which can at thesame time be a stabilizer and a tonicity agent is trehalose.

The “isoelectric point” or “pI” of a protein is the pH at which theprotein has a net overall charge equal to zero, i.e., the pH at whichthe protein has an equal number of positive and negative charges.Determination of the pI for any given protein can be done according towell-established techniques, such as, e.g., by isoelectric focusing.Isoelectric focusing is a technique for separating different moleculesby differences in their isoelectric point (pI). It is a type of zoneelectrophoresis, usually performed on proteins in a gel that takesadvantage of the fact that overall charge on the molecule of interest isa function of the pH of its surroundings.

Various aspects of the disclosure are described in further detail below.Additional definitions are set out throughout the specification.

Pharmaceutical Formulations

In some embodiments, the present disclosure provides a pharmaceuticalcomposition comprising an anti-Cx43 antibody, or antigen bindingfragment thereof, as described herein. The anti-Cx43 antibody, orantigen binding fragment thereof, can have a first, second and thirdheavy chain complementarity determining region (CDR) sequence having theamino acid sequence of SEQ ID NOs: 1, 2, and 3, respectively; and afirst, second and third light chain CDR sequence having the amino acidsequence of SEQ ID NOs: 4, 5, and 6, respectively.

In some embodiments, the anti-Cx43 antibody or antigen binding fragmentthereof can include a heavy chain variable domain having the amino acidsequence of SEQ ID NO: 7, and a light chain variable domain having theamino acid sequence of SEQ ID NO: 8.

In certain embodiments, the anti-Cx43 antibody or antigen bindingfragment thereof comprises a heavy chain having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9-17, and a lightchain having the amino acid sequence of SEQ ID NO: 18.

In certain embodiments, the anti-Cx43 antibody or antigen bindingfragment thereof binds to an epitope located within the amino acidsequence of FLSRPTEKTI (SEQ ID NO: 19).

In various embodiments, the anti-Cx43 antibody or antigen bindingfragment thereof can be formulated in pharmaceutically acceptableamounts and in pharmaceutically acceptable compositions. As used herein,“pharmaceutically acceptable” shall refer to that which is useful inpreparing a pharmaceutical composition that is generally safe,non-toxic, and neither biologically nor otherwise undesirable andincludes that which is acceptable for veterinary use as well as humanpharmaceutical use. Examples of “pharmaceutically acceptable liquidcarriers” include water and organic solvents. Preferred pharmaceuticallyacceptable aqueous liquids include PBS, saline, and dextrose solutionsetc.

As used herein, the term “pharmaceutically acceptable salt” means anypharmaceutically acceptable salt of the compounds disclosed herein. Forexample, pharmaceutically acceptable salts of any of the compoundsdescribed herein include those that are within the scope of soundmedical judgment, suitable for use in contact with the tissues of humansand animals without undue toxicity, irritation, allergic response andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example,pharmaceutically acceptable salts are described in: Berge et al., J.Pharmaceutical Sciences 66: 1-19, 1977 and in Pharmaceutical Salts:Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth),Wiley-VCH, 2008. The salts can be prepared in situ during the finalisolation and purification of the compounds described herein orseparately by reacting a free base group with a suitable organic acid.

Various literature references are available to facilitate selection ofpharmaceutically acceptable carriers or excipients. See, e.g.,Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: NationalFormulary, Mack Publishing Company, Easton, Pa. (1984); Hardman et al.(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, NY; Weiner, Wang, W., Int. J. Pharm. 185: 129-188 (1999) andWang, W., Int. J. Pharm. 203: 1-60 (2000), and Kotkoskie (2000)Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.

In some embodiments, the antibody formulation can comprise a buffer(e.g., histidine, acetate, phosphate or citrate buffer), a surfactant(e.g., polysorbate), and/or a stabilizer agent (e.g., sucrose), etc. Insome certain embodiments, the buffer can be selected from acetate/sodiumacetate, histidine/aspartic acid, citric acid/sodium citrate, dibasicsodium phosphate/sodium dihydrogen phosphate, and histidine/histidinehydrochloride. In certain embodiments, the buffer is histidine/asparticacid or histidine/histidine hydrochloride. In certain embodiments, thebuffer is histidine/histidine hydrochloride. In some embodiments, thesurfactant is polysorbate 80 (PS80). In certain embodiments, thestabilizer is selected from ethylenediaminetetraacetic acid (EDTA),sodium chloride, sorbitol, glycine, and sucrose. In certain embodiments,the stabilizer is sucrose.

In some embodiments, the antibody formulation can comprisepharmaceutically acceptable carriers, including, e.g., ion exchangers,alumina, aluminum stearate, lecithin, serum proteins, such as humanserum albumin, buffer substances such as phosphates, sucrose, glycine,sorbic acid, potassium sorbate, partial glyceride mixtures of saturatedvegetable fatty acids, water, salts or electrolytes, such as protaminesulfate, disodium hydrogen phosphate, potassium hydrogen phosphate,sodium chloride, zinc salts, colloidal silica, magnesium trisilicate,polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol,sodium carboxymethylcellulose, polyacrylates,polyethylene-polyoxypropylene-block polymers, and polyethylene glycol.In some embodiments, the antibody formulation further comprises asurfactant. In some embodiments, the surfactant is selected from thegroup consisting of polysorbate, sodium dodecyl sulfate, and nonionicsurfactant.

The formulation according to the disclosure can be in a liquid form, ina lyophilized form or in a liquid form reconstituted from a lyophilizedform. In certain embodiments, the formulation is in a liquid form. Theterm “liquid” as used herein in connection with the formulationaccording to the disclosure denotes a formulation which is liquid at atemperature of at least about 2 to about 8° C. under atmosphericpressure. The term “lyophilized” as used herein in connection with theformulation according to the disclosure denotes a formulation which ismanufactured by freeze-drying methods known in the art per se. Thesolvent (e.g., water) is removed by freezing followed by sublimation ofthe ice under vacuum and desorption of residual water at elevatedtemperature. The lyophilizate usually has a residual moisture of about0.1 to 5% (w/w) and is present as a powder or a physically stable cake.The lyophilizate is characterized by a fast dissolution after additionof a reconstitution medium.

The term “reconstituted form” as used herein in connection with theformulation according to the disclosure denotes a formulation which islyophilized and re-dissolved by addition of reconstitution medium.Suitable reconstitution media comprise but are not limited to water forinjection (WFI), bacteriostatic water for injection (BWFI), sodiumchloride solutions (e.g. 0.9% (w/v) NaCl), glucose solutions (e.g. 5%glucose), surfactant-containing solutions (e.g. 0.02% polysorbate 80),pH-buffered solutions (eg. phosphate-buffered solutions).

The formulation according to the disclosure is physiologically welltolerated, can be prepared easily, can be dispensed precisely and isstable with respect to decomposition products and aggregates over theduration of storage, during repeated freezing and thawing cycles andmechanical stress. It is stable at storage temperatures (e.g., −40° C.or −20° C. or 2-8° C.) over a period of more than 1 year.

The antibody formulations of the present disclosure can be an aqueoussolution. In some embodiments, the antibody formulation has not beensubjected to freezing temperatures, and/or have not been frozen, i.e.,they have remained in a liquid state. In some embodiments, the antibodyin the antibody formulation has not been subjected to lyophilization.

In some embodiments, the antibody formulations can have improvedstability. As used herein, the term “stability” generally is related tomaintaining the integrity or to minimizing the degradation,denaturation, aggregation or unfolding of a biologically active agentsuch as a protein, peptide or another bioactive macromolecule. As usedherein, “improved stability” generally means that, under conditionsknown to result in degradation, denaturation, aggregation or unfolding,the protein (e.g., antibody such as anti-Cx43 Ab), peptide or anotherbioactive macromolecule of interest maintains greater stability comparedto a control protein, peptide or another bioactive macromolecule.

In some embodiments, stability refers to an antibody formulation havinglow to undetectable levels of particle formation. The phrase “low toundetectable levels of particle formation” as used herein refers tosamples containing less than 30 particles/mL, less than 20 particles/mL,less than 20 particles/mL, less than 15 particles/mL, less than 10particles/mL, less than 5 particles/mL, less than 2 particles/mL or lessthan 1 particle/mL as determined by HIAC analysis or visual analysis. Insome embodiments, no particles in the antibody formulation are detected,either by HIAC analysis or visual analysis.

In some embodiments, stability refers to reduced fragmentation of theantibody. The term “low to undetectable levels of fragmentation” as usedherein refers to samples containing equal to or more than 80%, 85%, 90%,95%, 98% or 99% of the total protein, for example, in a single peak asdetermined by HPSEC, or in two peaks (e.g., heavy- and light-chains) (oras many peaks as there are subunits) by reduced Capillary GelElectrophoresis (rCGE), representing the non-degraded antibody or anon-degraded fragment thereof, and containing no other single peakshaving more than 5%, more than 4%, more than 3%, more than 2%, more than1%, or more than 0.5% of the total protein in each. The term “reducedCapillary Gel Electrophoresis” as used herein refers to capillary gelelectrophoresis under reducing conditions sufficient to reduce disulfidebonds in an antibody.

One of skill in the art will appreciate that stability of a protein isdependent on other features in addition to the composition of theformulation. For example, stability can be affected by temperature,pressure, humidity, pH, and external forms of radiation. Thus, unlessotherwise specified, stability referred to herein is considered to bemeasured at −20° C., one atmosphere pressure, 50% relative humidity, pHof 5.5, and normal background levels of radiation. Stability of theantibody in the antibody formulation can be determined by various means.In some embodiments, the antibody stability is determined by sizeexclusion chromatography (SEC). SEC separates analytes (e.g.,macromolecules such as proteins and antibodies) on the basis of acombination of their hydrodynamic size, diffusion coefficient, andsurface properties. Thus, for example, SEC can separate antibodies intheir natural three-dimensional conformation from antibodies in variousstates of denaturation, and/or antibodies that have been degraded. InSEC, the stationary phase is generally composed of inert particlespacked into a dense three-dimensional matrix within a glass or steelcolumn. The mobile phase can be pure water, an aqueous buffer, anorganic solvent, mixtures of these, or other solvents. Thestationary-phase particles have small pores and/or channels which willonly allow species below a certain size to enter. Large particles aretherefore excluded from these pores and channels, but the smallerparticles are removed from the flowing mobile phase. The time particlesspend immobilized in the stationary-phase pores depends, in part, on howfar into the pores they can penetrate. Their removal from the mobilephase flow causes them to take longer to elute from the column andresults in a separation between the particles based on differences intheir size.

In some embodiments, SEC is combined with an identification technique toidentify or characterize proteins, or fragments thereof. Proteinidentification and characterization can be accomplished by varioustechniques, including but not limited chromatographic techniques, e.g.,high-performance liquid chromatography (HPLC), immunoassays,electrophoresis, ultra-violet/visible/infrared spectroscopy, ramanspectroscopy, surface enhanced raman spectroscopy, mass spectroscopy,gas chromatography, static light scattering (SLS), Fourier TransformInfrared Spectroscopy (FTIR), circular dichroism (CD), urea-inducedprotein unfolding techniques, intrinsic tryptophan fluorescence,differential scanning calorimetry, and/or ANS protein binding.

In some embodiments, protein identification is achieved by high-pressureliquid chromatography. Various instruments, and apparatuses are known tothose of skill in the art to perform HPLC. Generally, HPLC involvesloading a liquid solvent containing the protein of interest onto aseparation column, in which the separation occurs. The HPLC separationcolumn is filled with solid particles (e.g. silica, polymers, orsorbents), and the sample mixture is separated into compounds as itinteracts with the column particles. HPLC separation is influenced bythe liquid solvent's condition (e.g. pressure, temperature), chemicalinteractions between the sample mixture and the liquid solvent (e.g.hydrophobicity, protonation, etc.), and chemical interactions betweenthe sample mixture and the solid particles packed inside of theseparation column (e.g. ligand affinity, ion exchange, etc.).

In some embodiments, the SEC and protein identification occurs withinthe same apparatus, or simultaneously. For example, SEC and HPLC can becombined, often referred to as SE-HPLC.

In some embodiments, the aqueous formulation comprises about 2 mg/mL toabout 100 mg/mL antibody wherein the antibody comprises a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3sequences of SEQ ID NOs: 1-3, and wherein the light chain variableregion comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQID NOs: 4-6, wherein said formulation is stable upon storage at about40° C. for at least 1 month. In some embodiments, the formulation isstable upon storage at about 25° C. for at least 3 months. In someembodiments, the formulation is stable upon storage at about 5° C. forat least 6 months. In some embodiments, the formulation is stable uponstorage at about 5° C. for at least 12 months. In some embodiments, theformulation is stable upon storage at about 5° C. for at least 18months. In some embodiments, the formulation is stable upon storage atabout 5° C. for at least 24 months, or 36 months.

The term “stable” can be relative and not absolute. Thus, in someembodiments the antibody is stable if less than 20%, less than 15%, lessthan 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored −20° C. for 6 months. In some embodiments, theantibody is stable if less than 20%, less than 15%, less than 10%, lessthan 5% or less than 2% of the antibody is degraded, denatured,aggregated or unfolded as determined by SEC HPLC when the antibody isstored at −20° C. for 12 months. In some embodiments, the antibody inthe antibody formulation is stable if less than 20%, less than 15%, lessthan 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at −20° C. for 18 months. In some embodiments, theantibody in the antibody formulation is stable if less than 20%, lessthan 15%, less than 10%, less than 5% or less than 2% of the antibody isdegraded, denatured, aggregated or unfolded as determined by SEC HPLCwhen the antibody is stored at −20° C. for 24 months.

In some embodiments, the antibody is stable if less than 20%, less than15%, less than 10%, less than 5% or less than 2% of the antibody isdegraded, denatured, aggregated or unfolded as determined by SEC HPLCwhen the antibody is stored at 23° C. to 27° C. for 3 months. In someembodiments, the antibody is stable if less than 20%, less than 15%,less than 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at 23° C. to 27° C. for 6 months. In someembodiments, the antibody is stable if less than 20%, less than 15%,less than 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at 23° C. to 27° C. for 12 months. In someembodiments, the antibody is stable if less than 20%, less than 15%,less than 10%, less than 5% or less than 2% of the antibody is degraded,denatured, aggregated or unfolded as determined by SEC HPLC when theantibody is stored at 23° C. to 27° C. for 24 months.

In some embodiments the antibody is stable if less than 6%, less than4%, less than 3%, less than 2% or less than 1% of the antibody isdegraded, denatured, aggregated or unfolded per month as determined bySEC HPLC when the antibody is stored at 40° C. In some embodiments theantibody is stable if less than 6%, less than 4%, less than 3%, lessthan 2% or less than 1% of the antibody is degraded, denatured,aggregated or unfolded per month as determined by SEC HPLC when theantibody is stored at 5° C.

In some embodiments, the antibody formulations of the present disclosurecan be considered stable if the antibody exhibits very little to no lossof the binding activity of the antibody (including antibody fragmentsthereof) of the formulation compared to a reference antibody as measuredby antibody binding assays know to those in the art, such as, e.g.,ELISAs, etc., over a period of 8 weeks, 4 months, 6 months, 9 months, 12months or 24 months. In some embodiments, the antibody stored at about40° C. for at least 1 month retains at least 60%, at least 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 98%,or at least about 99% of binding ability to Cx43 compared to a referenceantibody which has not been stored. In some embodiments, the antibodystored at about 5° C. for at least 6 months retains at least 80%, atleast about 85%, at least about 90%, at least about 95%, at least about98%, or at least about 99% of binding ability to Cx43 compared to areference antibody which has not been stored. In some embodiments, theantibody stored at about 40° C. for at least 1 month retains at least95% of binding ability to Cx43 compared to a reference antibody whichhas not been stored. In some embodiments, the antibody stored at about5° C. for at least 6 months retains at least 95% of binding ability toCx43 compared to a reference antibody which has not been stored.

The antibody formulations can provide low to undetectable levels ofaggregation of the antibody. The phrase “low to undetectable levels ofaggregation” as used herein refers to samples containing no more thanabout 5%, no more than about 4%, no more than about 3%, no more thanabout 2%, no more than about 1% and no more than about 0.5% aggregationby weight of protein as measured by high performance size exclusionchromatography (HPSEC) or static light scattering (SLS) techniques. Insome embodiments, less than 2% of the antibody forms an aggregate uponstorage at about 40° C. for at least 4 weeks as determined by asdetermined by HPSEC. In some embodiments, less than 2% of the antibodyforms an aggregate upon storage at about 5° for at least 3 months, atleast 6 months, at least 9 months, at least 12 months, at least 15months, at least 18 months, at least 24 months, or at least 36 months asdetermined by HPSEC.

It has been discovered herein the antibody formulations provided hereinresult in greatly reduced particle formation as determined by visualinspection, micro-flowing imaging (MFI), or size-exclusionchromatography (SEC). In some embodiments, the formulation issubstantially free of particles upon storage at about 40° C. for atleast 1 month as determined by visual inspection. In some embodiments,the formulation is substantially free from particles upon storage atabout 5° C. for at least 6 months, at least 9 months, at least 12months, at least 15 months, at least 18 months, at least 24 months, orat least 36 months as determined by visual inspection.

The formulations may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, and by the inclusion of various antibacterial and antifungalagents, e.g. paraben, chlorobutanol, phenol, sorbic acid, and the like.Preservatives are generally used in an amount of about 0.001 to about 2%(w/v). Preservatives comprise but are not limited to ethanol, benzylalcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens,benzalkonium chloride.

The antibody formulations described herein can have various viscosities.Methods of measuring viscosity of antibody formulations are known tothose in the art, and can include, e.g., a rheometer (e.g., Anton PaarMCR301 Rheometer with either a 50 mm, 40 mm or 20 mm plate accessory).In some embodiments of the present disclosure, the viscosities werereported at a high shear limit of 1000 per second shear rate. In someembodiments, the antibody formulation has a viscosity of less than 20centipoise (cP), less than 18 cP, less than 15 cP, less than 13 cP, orless than 11 cP. In some embodiments, the antibody formulation has aviscosity of less than 13 cP. One of skill in the art will appreciatethat viscosity is dependent on temperature, thus, unless otherwisespecified, the viscosities provided herein are measured at 25° C. unlessotherwise specified.

The antibody formulations can have different osmolarity concentrations.Methods of measuring osmolarity of antibody formulations are known tothose in the art, and can include, e.g., an osmometer (e.g., an AdvancedInstrument Inc 2020 freezing point depression osmometer). In someembodiments, the formulation has an osmolarity of between 200 and 600mosm/kg, between 260 and 500 mosm/kg, or between 300 and 450 mosm/kg.

The antibody formulation of the present disclosure can have various pHlevels. In some embodiments, the pH of the antibody formulation isbetween 4 and 7, between 4.5 and 6.5, between 5 and 6, or between 5.4 to5.6. In some embodiments, the pH of the antibody formulation is 5.5. Insome embodiments, the pH of the antibody formulation is 6.0. In someembodiments, the pH of the antibody formulation is 7.0. Various meansmay be utilized in achieving the desired pH level, including, but notlimited to the addition of the appropriate buffer.

In some embodiments, the antibody formulation can include: about 40-60mg/mL, preferably about 50 mg/mL of an anti-Cx43 antibody or antigenbinding fragment thereof; about 10-40 mM, preferably about 20 mMhistidine/histidine hydrochloride buffer; about 0.005%-0.05%, preferablyabout 0.02% w/v Polysorbate 80; and about 1%-20% w/v, preferably about8% w/v sucrose; wherein the formulation has a pH of between about 5.4 toabout 5.6, preferably about 5.5.

In some embodiments, the antibody formulation can include: about 50mg/mL of an anti-Cx43 antibody or antigen binding fragment thereof,comprising a heavy chain having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 9-17, and comprising a light chainhaving the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 9 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 10 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 11 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 12 and a lightchain having the amino acid sequence of SEQ ID

NO: 18; about 20 mM histidine/aspartic acid buffer; about 0.02% w/vPolysorbate 80; and about 8% w/v sucrose, wherein the formulation has apH of between about 5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 13 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 14 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 15 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 16 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In one embodiment, the antibody formulation can include: about 50 mg/mLof an anti-Cx43 antibody or antigen binding fragment thereof, comprisinga heavy chain having an amino acid sequence of SEQ ID NO: 17 and a lightchain having the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.

In some embodiments, the disclosure provides a kit comprising any of theantibody formulations described herein, the containers described herein,the unit dosage forms described herein, or the pre-filled syringedescribed herein.

Therapeutic Uses

In some embodiments, the antibody formulation of the present disclosurecan be used for pharmaceutical purposes. Antibodies used inpharmaceutical applications generally must have a high level of purity,especially in regard to contaminants from the cell culture, includingcellular protein contaminants, cellular DNA contaminants, viruses andother transmissible agents. See “WHO Requirements for the use of animalcells as in vitro substrates for the production of biologicals:Requirements for Biological Substances No. 50.” No. 878. Annex 1, 1998.In response to concerns about contaminants, The World HealthOrganization (WHO) established limits on the levels of variouscontaminants. For example, the WHO recommended a DNA limit of less than10 ng per dose for protein products. Likewise, the United States Foodand Drug Administration (FDA) set a DNA limit of less than or equal to0.5 pg/mg protein. Thus, in some embodiments, the present disclosure isdirected to antibody formulations meeting or exceeding contaminantlimits as defined by one or more governmental organizations, e.g., theUnited States Food and Drug Administration and/or the World HealthOrganization.

The antibody formulation of the present disclosure can be administeredto a subject through various means. In some embodiments, the antibodyformulation is suitable for parenteral administration, e.g., viainhalation (e.g., powder or aerosol spray), transmucosal, intravenous,subcutaneous, or intramuscular administration. In some embodiments, theformulation is an injectable formulation. In some embodiments, thedisclosure is directed to a sealed container comprising any of theantibody formulations as described herein.

In some aspects, the present disclosure is directed to variouspharmaceutical dosage forms. Various dosage forms could be applicable tothe formulations provided herein. See, e.g., Pharmaceutical Dosage Form:Parenteral Medications, Volume 1, 2′ Edition. In one embodiment, apharmaceutical unit dosage of the disclosure comprises the antibodyformulation in a suitable container, e.g. a vial or syringe. In oneembodiment, a pharmaceutical unit dosage of the disclosure comprises anintravenously, subcutaneously, or intramuscularly delivered antibodyformulation. In another embodiment, a pharmaceutical unit dosage of thedisclosure comprises aerosol delivered antibody formulation. In aspecific embodiment, a pharmaceutical unit dosage of the disclosurecomprises a subcutaneously delivered antibody formulation. In anotherembodiment, a pharmaceutical unit dosage of the disclosure comprises anaerosol delivered antibody formulation. In a further embodiment, apharmaceutical unit dosage of the disclosure comprises an intranasallyadministered antibody formulation.

A composition of the present disclosure can be administered by a varietyof methods known in the art. As will be appreciated by the skilledartisan, the route and/or mode of administration will vary dependingupon the desired results.

To administer a composition of the disclosure by certain routes ofadministration, it may be necessary to dilute the composition in adiluent. Pharmaceutically acceptable diluents include saline, glucose,Ringer and aqueous buffer solutions.

In a particular embodiment, the formulation according to the disclosureis administered by intravenous (i.v.), subcutaneous (s.c.) or any otherparental administration means such as those known in the pharmaceuticalart.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration, usually by injection, and include, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

The composition must be sterile and fluid to the extent that thecomposition is deliverable by syringe or an infusion system. In additionto water, the carrier can be an isotonic buffered saline solution,ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof.

The formulation according to the disclosure can be prepared by methodsknown in the art, e.g., ultrafiltration-diafiltration, dialysis,addition and mixing, lyophilisation, reconstitution, and combinationsthereof. Examples of preparations of formulations according to thedisclosure can be found hereinafter.

The pharmaceutical composition as described herein may be used intreatment of inflammatory disorders, including sterile as well asinfectious inflammation, such as inflammatory lung diseases,osteoarthritis, and spinal cord injury.

Sterile inflammation is a common event, triggered by physical, chemicalor metabolic noxiae. The different noxiae cause cell stress and hencestress responses. Many types of stress response exist (e.g. unfoldingprotein response, integrated stress response, oxidative stress), oftenentangled among each other. Stress responses trigger inflammation. Whennoxiae persist, inflammation does not resolve, resulting in a viciouscircle that has a key role in the pathophysiology of many humandisorders, including cancer, metabolic and genetic diseases.

The acute conditions that result from sterile inflammation includeischemia reperfusion injury (IRI), trauma (e.g., spinal cord injury,traumatic brain injury, peripheral nerve injury), crystal-inducedinflammation, and toxin exposure. Acute myocardial infarctions, cerebralinfarctions, acute kidney injury and solid organ transplantation are allconditions in which IRI occurs. Crystal deposition within joints leadsto gouty arthritis and elicits the classic clinical signs ofinflammation including redness, pain, heat, swelling and loss offunction. Toxins, such as acetaminophen or cobra venom, induce hepaticand muscle injury, respectively. Trauma, including crush injury,triggers an abrupt inflammatory response, and endogenous and microbialtriggers (from bacterial exposure) may contribute to inflammation inthis context.

Chronic conditions that trigger or result from sterile inflammationinclude particle-induced lung diseases such as asbestosis and silicosis,chronic pulmonary diseases such as cystic fibrosis and idiopathicpulmonary fibrosis, cardiovascular diseases such as atherosclerosis,some causes of chronic heart failure, certain cases of tumors, arthritis(e.g., osteoarthritis and rheumatoid arthritis (RA)), and autoimmuneconditions.

Infectious inflammation can be caused by various pathogens such asbacteria and fungi in a number of tissues.

Inflammatory diseases as used herein refer to a vast array of disordersand conditions that are characterized by inflammation. Examples includearthritis, allergy, asthma, autoimmune diseases, coeliac disease,glomerulonephritis, hepatitis, inflammatory bowel disease (includingCrohn's disease and Ulcerative Colitis), reperfusion injury andtransplant rejection.

Autoimmune diseases are diseases in which the immune system attacks itsown proteins, cells, and tissues, or in which immune effector T cellsare autoreactive to endogenous self peptides and cause destruction oftissue. Thus, an immune response is mounted against a subject's ownantigens, referred to as self antigens. A comprehensive listing andreview of autoimmune diseases can be found in The Autoimmune Diseases(Rose and Mackay, 2014, Academic Press). Autoimmune diseases include butare not limited to rheumatoid arthritis, Crohn's disease, Type 1diabetes, alopecia, multiple sclerosis, lupus, systemic lupuserythematosus (SLE), autoimmune encephalomyelitis, myasthenia gravis(MG), Hashimoto's thyroiditis, Goodpasture's syndrome, pemphigus (e.g.,pemphigus vulgaris), Grave's disease, autoimmune hemolytic anemia,autoimmune thrombocytopenic purpura, scleroderma with anti-collagenantibodies, mixed connective tissue disease, polymyositis, perniciousanemia, idiopathic Addison's disease, autoimmune-associated infertility,glomerulonephritis (e.g., crescentic glomerulonephritis, proliferativeglomerulonephritis), bullous pemphigoid, Sjogren's syndrome, insulinresistance, and autoimmune diabetes mellitus.

Neurodegenerative diseases are diseases such as Alzheimer's disease(AD), lysosomal storage disorders, bacterial meningitis, amyotrophiclateral sclerosis, hypoxia, ischemia, glaucoma, schizophrenia, majordepression, bipolar disorder, epilepsy, traumatic brain injury,post-traumatic stress disorder, Parkinson's disease, Down syndrome,spinocerebellar ataxia, Huntington's disease, radiation therapy inducedneurodegeneration, chronic stress induced neurodegeneration, andneurodegeneration associated with normal aging or abuse of neuro-activedrugs (such as alcohol, opiates, methamphetamine, phencyclidine, andcocaine).

Osteoarthritis is a type of joint disease that results from thebreakdown of joint cartilage and underlying bone. The most commonsymptoms are joint pain and stiffness, which can progress slowly overyears. Osteoarthritis is believed to be caused by mechanical stress onthe joint and low grade inflammatory processes. Damage from mechanicalstress with insufficient self repair by joints is believed to be theprimary cause of osteoarthritis.

Spinal cord injury is damage to the spinal cord that causes temporary orpermanent changes in its function. Symptoms may include loss of musclefunction, sensation, or autonomic function in the parts of the bodyserved by the spinal cord below the level of the injury. Injury canoccur at any level of the spinal cord and can be complete injury, with atotal loss of sensation and muscle function, or incomplete, meaning somenervous signals are able to travel past the injured area of the cord.Depending on the location and severity of the damage, the symptoms vary,from numbness to paralysis to incontinence. Long term outcomes alsorange widely, from full recovery to permanent tetraplegia (also calledquadriplegia) or paraplegia. Complications can include muscle atrophy,pressure sores, infections, and breathing problems.

Spinal cord injury can be traumatic or nontraumatic, and can beclassified into three types based on cause: mechanical forces, toxic,and ischemic (from lack of blood flow). The damage can also be dividedinto primary and secondary injury: the cell death that occursimmediately in the original injury, and biochemical cascades that areinitiated by the original insult and cause further tissue damage. Thesesecondary injury pathways include the ischemic cascade, inflammation,swelling, cell suicide, and neurotransmitter imbalances. They can takeplace for minutes or weeks following the injury.

EXAMPLES

The following examples are presented so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the compositions and methods and are not intended to limitthe scope of what the inventors regard as their invention.

Example 1: Materials and Methods Abbreviations

Abbreviation Full name Caliper-NR/CE-NR Non-Reduced CE-SDS CaliperCaliper-R/CE-R Reduced CE-SDS Caliper CE-SDS CapillaryElectrophoresis-Sodium Dodecyl Sulfate CEX Cation ExchangeChromatography cIEF Capillary Isoelectric Focusing DS Drug substance DPDrug product ECD Equivalent Circular Diameter FT Freeze/Thaw HMW HighMolecular Weight LMW Low Molecular Weight mDSC Modulated DifferentialScanning Calorimetry MFI Micro Flowing Imaging/Microfluidic Imaging mMMillimoles/Liter MW Molecular Weight NA Not Applicable ND Not DetectedPh. Eur. European Pharmacopoeia pI Isoelectric Point PS80 Polysorbate 80rpm Round Per Minute/Revolution Per Minute RT Room TemperatureSDS-Caliper Caliper-Sodium Dodecyl Sulfate SDS-CE-R Reduced CapillaryElectrophoresis-Sodium Dodecyl Sulfate SDS-CE-NR Non-Reduced CapillaryElectrophoresis-Sodium Dodecyl Sulfate SEC-HPLC Size Exclusion HighPerformance Liquid Chromatography USP United States Pharmacopoeia w/vWeight/Volume A Agitation C Cycle D Day M Month T0 Time 0 W Week

Equipment

Description Vendor Model Agilent HPLC Agilent Technologies 1260 seriesSingapore (Sales)Pt (1260/1290) Automated Bioanalysis PerkinElmerLabChip ® GXII System Touch HT Capillary Isoelectric ProteinSimple iCE3or equivalent Focusing Analyzer Centrifuge Eppendorf Centrifuge 5804RClarity Detector Tianda Tianfa YB-2 Differential scanning MalvernMicrocal VP-Capillary calorimetry Drug Storage Box Haier HYC-940Electronic Balance Mettler Toledo MS6002S/0/MS1003S/ 01/XS205 MFIProteinSimple 5200 Modulated Differential TA Instruments-Waters DSCQ2000 Scanning Calorimetry LLC Osmometer Advanced Instruments. Advanced2020 INC pH Meter Mettler Toledo S40 Refrigerator HaierHYC-940/DW-40L508 Refrigerator Eppendorf U725 Safety Hood ShanghaiShangjing BSC-II-A2 Safety Hood Sujing Sutai BSC-II-A2 Thermostat ShakerShanghai Tiancheng TS-200B Stability Chamber MMM Climacell 707 Ultra-lowTemperature Eppendorf U725 Freezer UV spectrophotometer ThermoScientific NanoDrop 2000

Reagents

Reagent Grade Vendor Catalog # Lot# L-Histidine Multi-Compendial J. T.Baker 2080-06 0000090914 L-Histidine- Multi-Compendial J. T. Baker2081-06 0000179922 monohydrochloride Aspartic acid ph Eur/USP AppliChemA1701,1000 6T012474 Sodium dihydrogen ph Eur/BP/USP/JPE/E339 Merck1.06345.9026 K93518945 phosphate dihydrate Di-sodium hydrogen phEur/BP/USP Merck 1.06576.9029 K45710476 phosphate dihydrate Citric Acidph Eur/BP/JP/USP/E330 Merck 1.00242.5000 K48745442711 MonohydrateTri-Sodium Citrate ph Eur/BP/JP/USP/E331 Merck 1.06432.5000 K93697932Dihydrate Acetic Acid EP/BP/JP/USP J T Baker 9526-03 0000084970 SodiumAcetate, bio ph Eur/BP/JP/USP Merck 1.37012.9029 AM1027312 TrihydrateEDTA USP J. T. Baker 8995-01 0000172864 NaCl EP/BP/USP/JP Merck1.16224.5000 K47447424 Polysorbate 80 Multi-Compendial NOF NA 704352ASucrose Multi-Compendial Pfanstiehl S-124-1-MC 36920A Glycine CHPTianjin NA AGLY160124 Tianyao Sorbitol USP Merck 1.11597.2500 M852697705Description Vendor Catalog # Lot # 20 mL Ultrafiltration SartoriusStedim VS2022 1709032VS/1802014VS centrifuge tube 2 R Vial Schott(Suzhou) V002711080D 6104481548 6 R Vial Schott (Suzhou)V006111112C/1142196 6104358817 13 mm Rubber Stopper West (U.S.)1970-0004 D000063205 20 mm Rubber Stopper West (Singapore) 7002-23543172022309 13 mm Plastic-aluminum West (U.S.) 5413-0921 0000928228 cap20 mm Plastic-aluminum West (India) 5420-3627 00001235077 cap

These anti-Cx43 Ab formulation development studies were aimed to developstable liquid formulations that support the long-term storage of theanti-Cx43 Ab drug product, including stabilizers, surfactants andosmolality regulators in a stable pH/buffer system. The studies includedpH/Buffer screening, excipient screening and PS80 strength screening.The impact of the buffer system, pH, excipients and PS80 on productstability was evaluated under freeze/thaw, agitation and heat stressconditions.

The target concentration of the anti-Cx43 Ab was 50 mg/mL, which wasused for these formulation studies. Based on the results of thepH/Buffer screening study, 20 mM histidine/histidine hydrochloridebuffer at pH 5.5 was deemed the appropriate pH/buffer system for thefurther formulation studies.

The excipients and PS80 strength screening studies showed that theanti-Cx43 Ab in histidine buffer with sucrose was relatively more stablethan that with sodium chloride, sorbitol or glycine. The addition ofPS80 significantly improved the stability of the anti-Cx43 Ab at anoptimal concentration of 0.02%, while the EDTA and protein concentrationstudies showed that they provided no significant effect in the stabilityof the anti-Cx43 Ab.

50 mg/mL anti-Cx43 Ab in 20 mM histidine/histidine hydrochloride at pH5.5 with 8% sucrose and 0.02% (w/v) PS80 was selected for theformulation confirmation study.

Sample Number Management Rules Sample Number: PPP-YYYYMMNN-X-CC-TT

PPP represents the numerical part of the project name (this project is2144). YYYY, MM and NN represents the year, the month and the serialnumber of sample preparation in this month, respectively.

X represents the testing condition. For example, FT and A representsfreeze-thaw and agitation, respectively.

CC represents the testing temperature. For example, 05, 25 and 40represents 2˜8° C., 25° C. and 40° C., respectively.

TT represents the testing time. For example, T0, 7D, 4W and 1Mrepresents the start time, 7 days, 4 weeks and 1 months, respectively.

F represents the formulation number. For example, F1 and F2 representsformulation 1 and formulation 2, respectively.

For instance: 2144-20180601-25-4W represents the first sample of projectanti-Cx43 Ab prepared in June 2018. The sample was stored upright at 25°C. for 4 weeks.

Analytical Methods Appearance

The appearance of samples, including clarity, color, and visibleparticles, was examined against a black and white background using aYB-2 light box.

pH

The pH was measured using a Mettler Toledo S40 pH Meter. The pH meterwas calibrated prior to use.

Osmolality

Osmolality was measured using an Advanced 2020 Multi-Sample Osmometerusing 20 μL of sample. The testing accuracy of the osmometer wasconfirmed with a 290 mOsmol/kg reference.

MFI

A Microflow Imaging (MFI) system was used for sub-visible particleanalysis. According to the user's manual, the MFI test was performedwith more than 1.3 mL samples. The MFI data was analyzed with the MVASsoftware. The final data was reported as the total particle number atdifferent size ranges.

Protein Concentration

The ultraviolet absorbance of a protein solution depends on theabsorption properties of the aromatic amino acid residues of the proteinmolecule. According to the Beer-Lambert Law, the concentration of aprotein solution can be calculated based on its absorbance at a givenwavelength, the cuvette cell path length, and the extinction coefficientvalue. DropSense 96 has two path lengths of 0.1 mm and 0.7 mm, andchooses the appropriate path length automatically. The instrumentobtained the absorbance value of anti-Cx43 Ab at 280 nm, factoring inthe appropriate path length. Therefore, the protein concentration wascalculated as the absorbance value divided by 1.420, the extinctioncoefficient of the anti-Cx43 Ab. The relationship of the absorbancevalue (A) of the protein solution at a particular ultravioletwavelength, the protein concentration (c), optical path (b) andextinction coefficient (ε) were in accord with the following formula:A=ε*b*c (A is the absorbance value, ε is the absorbance coefficient, bis the optical path and c is the concentration). The extinctioncoefficient of anti-Cx43 Ab was 1.420 AU*mL*mg⁻¹*cm⁻¹. UV absorption at280 nm was measured using a Nanodrop 2000 spectrophotometer.

DSC

Differential scanning calorimetry (DSC) was utilized to measure thethermal stability of proteins by detecting the heat capacity of thesample in heat flow. Specifically, DSC was used to measure the thermaltransition midpoint (Tm) and onset of melting (Tmonset), which areindicators of the relative stability of the protein in solution. Sampleswere diluted to 1 mg/mL with a reference buffer. An aliquot of 400 μL ofreference buffer was added into each odd-numbered well of a 96-wellplate while an aliquot of 400 μL of each sample was added into thecorresponding even-numbered well. The scanning temperature ranged from20° C. to 100° C. with a scan rate of 200° C./hr. Data analysis wasperformed using MicroCal VP Capillary DSC Automated data analysissoftware 2.0.

cIEF

The method of Imaged Capillary Isoelectric focusing (iCIEF) separatesproteins based on their charge differences in a pH gradient. Under anexternal electric field, the charge variants of monoclonal antibodiesmigrate along a continuous pH gradient formed by ampholyte additives.The charge variant stops where the pH equals to its pI. The pI value andrelative abundance of the resolved peaks are identified and quantifiedwith software. The master mix was prepared with the following proportion(for one sample amount): 0.5 μL pI 7.40 marker; 0.5 μL pI 9.46 marker; 1μL Pharmalyte 3-10; 3 μL Pharmalyte 8-10; 35 μL 1% Methylcellulose; 40μL H₂O. The solution for one sample injection was composed of 20 μL of1.0 mg/mL diluted sample and 80 μL of master mix.

SDS-Caliper (Reduced and Non-Reduced)

SDS-Caliper is a high throughput chip based method which separatesproteins mainly by their molecular size. Before each sample was tested,pretreatment, such as incubation with sample buffer, SDS andN-ethylmaleimide (for non-reduced) or dithiothreitol (for reduced) at70° C. for 10 min was necessary. The loading mix with a minimum volumeof 42 μL (final protein concentration of 0.045 mg/mL) was then tested byLabChip GXII Touch at excitation/emission wavelengths of 635 and 700 nm.The final results were analyzed by Empower software.

SEC-HPLC

Size exclusion chromatography (SEC) is a purity analysis method thatseparates proteins based on their size. Following separation, therelative percentages of HMW species, monomer and LMW species arequantified via UV detection. SEC was performed as follows: if the samplewas above 10 mg/mL, it was diluted to 10 mg/mL with mobile phase beforeSEC analysis. 100 μg of sample was injected into an Agilent 1260 HPLCsystem equipped with a TSKgel G3000SWXL column (7.8×300 mm, 5 μmparticle size) and a UV detector (detection wavelength: 280 nm). Themobile phase was 50 mM phosphate buffer with 300 mM Sodium Chloride (pH6.8±0.1). An isocratic gradient was applied for 20 min at a flow rate of1 mL/min.

mDSC

Modulated Differential Scanning calorimetry (mDSC) was performed using aDSC-Q2000 system (TA instruments-Waters LLC). Tzero aluminum cruciblesand Tzero aluminum lids, all from TA instruments, were used to containthe sample to be measured and to seal the crucible by means of a Tzeropress. An empty Tzero crucible was similarly prepared and used as areference. Approximately 10 μL DS was added, pressed flat andtransferred in a Tzero crucible sealed with a Tzero lid by means of aTzero press. The calibration scanning program was equilibrated at−60.00° C. for 5 min, then was run at a constant temperature rate of5.00° C./min to 10.00° C. Data acquisition and processing were performedwith the help of Universal Analysis Software package.

Cation Exchange Chromatography (CEX)

CEX measures the charge heterogeneity of a monoclonal antibody solutionby separating proteins according to differences in their net chargenumber in a buffered solution. Samples in low salt buffer, at a pH belowthe isoelectric point have a net positive charge and adsorb on thechromatographic resin which is negatively charged. A pH gradient is usedto elute the different protein species based on charge heterogeneity,with the most positively charged species binding the strongest andtherefore requiring the higher pH. The different eluted charged speciesare detected by ultraviolet absorbance at 280 nm. The percentage of mainpeak, acid peak and basic peak of the samples are determined by themethod of peak area normalization. CEX was performed on an Agilent 1260series Infinity system and a ProPac WCX-10 column. The mobile phase Aused here was 16 mM 2-Methylpiperazine, 16 mM Imidazole, 16 mM Tris, pH5.0±0.1. The mobile phase B was 16 mM 2-Methylpiperazine, 16 mMImidazole, 16 mM Tris, 80 mM NaCl, pH=10.9±0.1. The flow rate was set as1 mL/min. Samples were diluted to 1 mg/mL with mobile phase A and 100 μLof samples were eluted by gradient increasing the amount of mobile phaseB. The detection wavelength was set at 280 nm. The running time was 60minutes.

CE-SDS (Reduced)

Reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) is apurity analysis method that separates proteins based on theirelectrophoretic mobility, where proteins of smaller sizes move fasterand larger sizes move slower. In this method, the diluted protein sampleis first denatured with SDS then reduced with p-Mercaptoethanol (BME)before being injected into an uncoated capillary filled with a viscousSDS gel solution. Components of different molecule sizes in the proteinsamples were detected as they passed through the capillary with PDAdetector at 220 nm.

CE-SDS (Non-Reduced)

Non-reduced Capillary Electrophoresis-Sodium Dodecyl Sulfate (CE-SDS) isa purity analysis method that separates proteins based on theirelectrophoretic mobility, where proteins of smaller sizes move fasterand larger sizes move slower. In this method, the diluted protein sampleis first alkylated by N-ethylmaleimide (NEM) to prevent thermallyinduced fragmentation, then denatured with SDS before being injectedinto an uncoated capillary filled with a viscous SDS gel solution.Components of different molecule sizes in the protein samples weredetected as they passed through the capillary with PDA detector at 220nm.

Potency Binding Antigen

anti-Cx43 Ab ELISA potency assay is an enzyme-linked immunosorbent assaywhere the anti-Cx43 Ab product binds to biotin-peptide coated onstreptavidin coated 96-well plates. Serially diluted anti-Cx43 Absamples and a reference standard are allowed to bind to biotin-peptideon the plate. After washing, horseradish peroxidase (HRP)-conjugatedgoat anti-human IgG is added to the wells allowing it to interact withthe anti-Cx43 Ab product captured during previous step. After a finalwash step, the 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate solutionis added to wells. TMB specifically reacts with the peroxide in thepresence of peroxidase and produces a colorimetric signal that isproportional to the amount of anti-Cx43 Ab product bound to the wells.The dose response curves are fitted using a 4PL model and the resultsare reported as Relative Potency using the EC₅₀ values of the ReferenceStandard (RS) vs. the Sample.

Example 2: pH/Buffer Screening

The pH/Buffer screening study was to determine the optimal pH/buffersystems for the anti-Cx43 Ab drug product formulation. The goal of thisstudy was to select one pH/buffer system with maximum stabilizingcapability for the anti-Cx43 Ab drug product for further formulationdevelopment studies.

Nine pH/buffer systems were designed based on the protein molecule andthe application of buffer systems. Ultra-filtration centrifugal devicewas used to perform buffer-exchange of anti-Cx43 Ab DS (Lot:2144SD180528K01Y01D01). The DS formulated in 20 mM histidine/histidinehydrochloride buffer at pH 5.5 was generated from a 50 L pool. Theanti-Cx43 Ab concentration in this study was 50 mg/mL. All formulationswere filtered and then distributed into 2R vials. Vials of eachformulation were stored at both 25±2° C. and 40±2° C. for up to 4 weeks.Samples were retrieved timely at each time point and kept at 2˜8° C.before analysis. Testing items including appearance, pH, Conc-UV280,SEC-HPLC, cIEF, Caliper-SDS(R&NR), DSC were performed for this study.The sampling plan is listed in Table 1.

TABLE 1 Study Parameters from the anti-Cx43 Ab pH/Buffer ScreeningStored at 25° C. Stored at 40° C. formulation Sample Buffer Time 4 4 No.No. System pH 0 2 W W(opt) 2 W W(opt) B1 2144- 20 mM 5.0 x, y, z x x, zx x, z 20180601 Acetate B2 2144- 20 mM 5.0 x, y, z x x, z x x, z20180602 Histidine/ B3 2144- Aspartic 5.5 x, y, z x x, z x x, z 20180603acid B4 2144- 20 mM 5.5 x, y, z x x, z x x, z 20180604 Citrate B5 2144-6.0 x, y, z x x, z x x, z 20180605 B6 2144- 20 mM 5.5 x, y, z x x, z xx, z 20180606 Histidine B7 2144- 6.0 x, y, z x x, z x x, z 20180607 B82144- 6.5 x, y, z x x, z x x, z 20180608 B9 2144- 20 mM 7.0 x, y, z x x,z x x, z 20180609 Phosphate Notes: x = Appearance; SEC-HPLC; cIEF;Caliper-SDS(R&NR); y = DSC; z = pH; Conc-UV280; (opt) = optional.

An ultra-filtration centrifugal device (30,000 MWCO PES, VIVASPIN 20)was used to perform buffer-exchange of anti-Cx43 Ab DS. The componentsof each final target formulation are shown in Table 1. Multiple roundsof ultrafiltration were performed until the exchange rate exceeded 98%.Then the protein concentration was adjusted to 50 mg/mL by thecorresponding formulation buffers. Each formulation was filtered througha 0.22 um filter (Millipore Express PES Membrane) and then distributedinto 2R vials with a 1 mL/vial filling volume. Vials were immediatelystoppered and sealed after filling. All the filtration, filling andsealing operations were conducted in a bio-safety hood. The appropriatenumber of vials for each formulation were placed in 25° C. and 40° C.stability chambers respectively. Samples were drawn and analyzed atpre-determined time points.

Thermograms of anti-Cx43 Ab in the different buffer systems are shown inFIG. 1. The Tm onset value, the temperature at which Abs start tounfold, is considered an indicator for the overall thermal stability ofthe formulation. As shown in Table 2, the B8 and B9 samples had lower TmOnset than the others. This indicated that the thermal stability ofanti-Cx43 Ab was not significantly influenced by other pH/buffer systemsexcept B8 and B9.

TABLE 2 DSC data from the anti-Cx43 Ab pH/Buffer screening studyFormulation Tm Onset Tm1 Tm2 Tm3 No. (° C.) (° C.) (° C.) (° C.) B1 62.871.1 79.3 86.5 B2 60.2 71.9 79.6 85.1 B3 62.0 71.4 79.5 86.0 B4 61.470.2 78.8 86.3 B5 62.4 70.2 78.3 86.5 B6 60.2 70.7 78.7 84.9 B7 62.271.0 79.1 86.6 B8 58.9 70.6 79.5 86.9 B9 59.9 69.4 76.2 86.5

The appearance, protein concentration and pH results of anti-Cx43 Ab indifferent buffer systems are summarized in Table 3 and Table 4.

The concentration of 9 samples were about 50 mg/mL, and the pH valueswere around the target pH. All the samples were colorless, slightlyopalescent and free of visible particles at T0, while the opalescentlevels of the B4, B5 and B9 samples were deeper than the others. After 2weeks of storage at 25±2° C. and 40±2° C., slightly visible particleswere found in all samples due to the absence of PS80.

This data suggested that the anti-Cx43 Ab was relatively more stable inthe B1, B2, B3, B6, B7 and B8 pH/buffer systems than other candidates.

TABLE 3 Protein concentration and pH results from the pH/Bufferscreening study Formulation Protein concentration mg/mL pH No. T0 25-4 W40-4 W T0 25-4 W 40-4 W B1 51.9 51.4 51.8 5.2 5.2 5.3 B2 49.7 49.3 49.55.2 5.3 5.3 B3 50.9 50.5 50.9 5.5 5.7 5.7 B4 51.0 50.9 50.6 5.5 5.5 5.5B5 50.5 50.1 50.1 5.9 6.0 5.9 B6 49.5 49.2 49.4 5.5 5.6 5.6 B7 49.3 49.548.9 6.0 6.1 6.2 B8 51.1 50.9 51.3 6.5 6.6 6.6 B9 51.0 51.2 50.7 7.0 6.96.9

TABLE 4 Appearance results from the pH/Buffer screening studyFormulation Appearance No. T0 25-2 W 25-4 W 40-2 W 40-4 W B1 A B B B BB2 A B B B B B3 A B B B B B4 A B B B B B5 A B B B B B6 A B B B B B7 A BB B B B8 A B B B B B9 A B B B B Notes: A = Colorless, slightlyopalescent and free of visible particles; B = Colorless, slightlyopalescent and slightly visible particles.

The SEC-HPLC results for all samples are shown in Table 5 and FIG. 2.All samples had comparable SEC purity with the main peak around 99.0% atT0. After incubation at 25° C. for 4 weeks, a slight decline of the SECmain peak in the range of 0.8%˜1.9% was observed. Sample B9, withphosphate buffer, showed a marginally higher decrease at 1.9%. Forsamples incubated at 40° C., a significant decrease of the main peak wasobserved after 2 weeks of storage. After incubation at 40° C. for 4weeks, the decline of the main peak was in the range of 2.1%˜4.2%.Differentiation between samples was not apparent except for the B4, B5and B9 samples. The purity decline in the B4, B5 and B9 samples were2.8%, 2.7% and 4.2% respectively. The SEC data indicated the anti-Cx43Ab was relatively more stable in B2, B3 and B6.

TABLE 5 SEC-HPLC results from the pH/Buffer screening study SEC-HPLCresults 25- 25- 40- 40- Formulation No. T0 2 W 4 W 2 W 4 W Main peak %B1 99.2 99.2 98.3 97.4 96.8 B2 99.3 99.3 98.5 97.6 97.0 B3 99.3 99.298.4 97.6 97.2 B4 99.2 98.9 98.1 97.0 96.4 B5 99.1 98.6 97.8 96.9 96.4B6 99.3 99.2 98.5 97.6 97.2 B7 99.3 99.1 98.4 97.6 96.9 B8 99.2 99.098.2 97.3 96.9 B9 98.9 97.9 97.0 95.8 94.7 HMW % B1 0.7 0.7 0.8 1.0 1.0B2 0.6 0.6 0.7 0.8 0.8 B3 0.7 0.7 0.8 0.8 0.9 B4 0.8 1.0 1.1 1.4 1.6 B50.8 1.3 1.4 1.6 1.8 B6 0.7 0.7 0.8 0.8 0.9 B7 0.7 0.8 0.8 0.9 1.0 B8 0.70.9 1.0 1.1 1.1 B9 1.1 2.0 2.1 2.6 3.1 LMW % B1 0.1 0.1 0.9 1.7 2.1 B20.1 0.1 0.8 1.7 2.2 B3 0.1 0.1 0.8 1.5 1.9 B4 0.1 0.1 0.8 1.6 2.0 B5 0.10.1 0.8 1.4 1.8 B6 0.1 0.1 0.8 1.6 1.9 B7 0.1 0.1 0.8 1.5 2.1 B8 0.1 0.10.8 1.7 2.0 B9 0.1 0.1 0.8 1.6 2.2

cIEF was used to determine the isoelectric point (pI) and charge variantdistribution of anti-Cx43 Ab. The cIEF results for all samples are shownin Table 6 and FIG. 3. There was no significant change in pI for all ofthe samples. The pI value of all samples was 8.7. After storage at 25±2°C. for 4 weeks, the main peak of almost all of the samples declinedslightly except for sample B2. The main peak decline for sample B9 was7.7%. After storage at 40±2° C. for 4 weeks, the main peak of eachformulation significantly declined, together with significantlyincreased acidic peaks. The main peaks of formulation B5, B8 and B9 weredecreased to 23.0%, 22.0% and 7.9% respectively. In contrast, the mainpeak declines for B2, B3 and B6 were relatively milder than that of theother samples. The cIEF data indicated that the anti-Cx43 Ab wasrelatively more stable in B2, B3 and B6.

TABLE 6 cIEF results from the pH/buffer screening study cIEF resultsFormulation 25- 25- 40- 40- Purity No. T0 2 W 4 W 2 W 4 W pI value B1-B98.7 8.7 8.7 8.7 8.7 Main peak % B1 46.3 46.8 44.1 35.2 25.5 B2 45.1 45.845.2 35.4 26.7 B3 45.8 45.5 44.5 35.5 26.4 B4 46.3 46.5 45.0 34.6 25.2B5 46.3 46.6 45.7 32.0 23.0 B6 46.2 44.7 44.0 36.0 27.3 B7 45.7 46.143.8 33.9 24.4 B8 45.1 44.7 41.3 31.9 22.0 B9 45.7 42.9 38.0 17.5 7.9Acidic peak % B1 40.6 39.1 42.6 51.1 62.0 B2 40.9 39.9 42.1 49.2 59.8 B341.1 41.0 42.3 51.8 63.6 B4 41.1 39.5 42.2 52.6 63.9 B5 41.4 39.5 42.055.6 66.6 B6 40.1 40.7 42.4 50.5 60.4 B7 41.0 40.9 43.4 53.9 65.2 B841.4 41.4 45.8 56.2 67.3 B9 41.3 44.1 49.1 73.2 87.5 Basic peak % B113.1 14.1 13.3 13.7 12.5 B2 14.1 14.2 12.7 15.3 13.5 B3 13.0 13.5 13.212.7 10.1 B4 12.6 14.0 12.9 12.8 11.0 B5 12.3 14.0 12.3 12.4 10.4 B613.6 14.6 13.6 13.5 12.3 B7 13.2 13.0 12.8 12.2 10.4 B8 13.5 13.9 12.911.9 10.7 B9 13.1 13.0 12.8 9.3 4.6

The SDS-Caliper results for all formulations are shown in Table 7, FIG.4 and FIG. 5.

There were no significant changes in non-reduced SDS-Caliper purity andreduced SDS-Caliper purity for all samples after storage at 25±2° C. for4 weeks. After 4-weeks of storage at 40±2° C., the non-reducedSDS-Caliper purity of samples B5 and B9 declined to 66.7% and 60.5%respectively, which were greater than other formulations. The main peakdeclines of samples B2, B3 and B6 were relatively milder than that ofother samples. The reduced SDS-Caliper purity of all samples declinedslightly except B5 and B9. The SDS-Caliper data indicated that theanti-Cx43 Ab was relatively more stable in B2, B3 and B6.

TABLE 7 SDS-Caliper results from the pH/Buffer screening studySDS-Caliper Purity Non-reduced SDS-Caliper Purity % Reduced SDS-CaliperPurity % Formulation 25- 25- 40- 40- 25- 25- 40- 40- No. T0 2 W 4 W 2 W4 W T0 2 W 4 W 2 W 4 W B1 98.5 97.5 97.9 89.2 78.8 98.1 97.9 97.9 96.194.6 B2 98.5 97.4 98.0 89.3 80.6 98.1 97.8 98.0 96.4 94.9 B3 98.6 97.597.8 84.6 75.8 98.1 97.6 97.7 95.8 93.2 B4 98.5 97.3 98.0 86.4 76.1 97.897.6 97.5 95.7 93.0 B5 98.3 97.1 97.8 80.7 66.7 97.8 97.4 97.3 94.3 89.0B6 98.6 97.5 97.9 85.7 74.7 98.0 97.7 97.7 95.8 93.6 B7 98.5 96.4 97.882.9 72.1 98.0 97.5 97.5 95.2 91.9 B8 98.4 96.9 97.8 83.9 77.1 97.9 97.797.6 94.3 93.2 B9 98.5 96.7 96.8 80.5 60.5 97.8 97.4 97.0 94.7 86.2

In this study, nine samples in varying pH/buffer systems were designedand incubated at 25±2° C. and 40±2° C. On the basis of all the results,the performance of formulation B6 (20 mM histidine/histidinehydrochloride buffer at pH 5.5) and B2 (20 mM histidine/aspartic acidbuffer at pH 5.0) were better than the other formulations. Inconclusion, 20 mM histidine/histidine hydrochloride buffer at pH 5.5(B6) would be used as a lead formulation and 20 mM histidine/asparticacid buffer at pH 5.0 (B2) would be used as a backup formulation forfurther studies.

Example 3: Excipients and PS80 Strength Screening

The aim of the excipients and PS80 screening study was to identify themost stabilizing excipients and evaluate the optimal strength of PS80for the anti-Cx43 Ab in candidate buffer systems.

20 mM histidine/histidine hydrochloride buffer at pH 5.5 (B6) was chosenas the buffer system, and combined with sodium chloride, sorbitol,glycine, sucrose, PS80, and EDTA according to the study plan. 20 mMhistidine/aspartic acid buffer at pH 5.0 (B2) was used as a backupbuffer for the excipients and PS80 strength screening study. Nineformulations were designed as shown in Table 8.

Formulations were frozen/thawed (˜40±5° C./RT) for 5 cycles, agitated at300 rpm at 25° C. for 7 days, and stored at 2˜8° C., 25±2° C. and 40±2°C. for 4 weeks. Samples were retrieved timely at each time point andkept at 2˜8° C. before analysis. Testing items including appearance, pH,Conc-UV280, osmolality, SEC-HPLC, cIEF, Caliper-SDS (R&NR) and MFI wereperformed for this study. Table 9 shows the sampling conditions for theexcipients and PS80 strength screening study.

TABLE 8 Formulation designed table from the anti-Cx43 Ab excipients andPS80 strength screening study Formulation Buffer Target PS 80 EDTA NaClSorbitol Glycine Sucrose No. Sample No. system concentration (w/v) (w/v)(mM) (mM) (mM) (w/v) F1 2144-20180801 H5.5 50 mg/mL 0.02% / 150 mM / / /F2 2144-20180802 / / 245 mM / / F3 2144-20180803 / / / 260 mM / F42144-20180804 / / / / 8% F5 2144-20180805 0.002% / / / 8% (0.068 mM) F62144-20180806 0.05% / / / / 8% F7 2144-20180807 / / / / / 8% F82144-20180808 25 mg/mL 0.02% 0.002% / / / 8% F9 2144-20180809 H-D-5.0 50mg/mL 0.02% 0.002% / / / 8% Notes: H5.5: 20 mM histidine/histidinehydrochloride buffer at pH 5.5; H-D-5.0: 20 mM histidine/aspartic acidbuffer at pH 5.0.

TABLE 9 Study parameters from the anti-Cx43 Ab excipients and PS80strength screening −40 ± 5° C./ 300 rpm 25 ° C. Formulation RIFreeze/Thaw Agitation 2~8° C. 25 ± 2° C. 40 ± 2° C. No. T0 5 C. 7 D 4 W(8 W) 2 W 4 W 2 W 4 W F1-F9 x, y x x x (x) x x x x Notes: x =Appearance, pH, SEC-HPLC, cIEF, MFI, Caliper; y = Conc-UV280,Osmolality; ( ) = optional.

Anti-Cx43 Ab DS (Lot: 2144SD180528K01Y01D01) formulated in 20 mMhistidine/histidine hydrochloride buffer at pH 5.5 was generated from a50 L pool. An ultra-filtration centrifugal device (30,000 MWCO PES,VIVASPIN 20) was used to perform buffer-exchange of anti-Cx43 Ab DS. Thecomponents of each final target formulation were calculated as describedin Table 1. Multiple rounds of ultrafiltration were performed until theexchange rate exceeded 98%. The protein concentration was then adjustedto 25 mg/mL (B8) and 50 mg/mL (except B8) using the correspondingformulation buffers. Each formulation was filtered through a 0.22 μmfilter (Millipore Express PES Membrane) and then distributed into 6Rvials with 4 mL/vial filling volume. Vials were immediately stopperedand sealed after filling. All the filtration, filling and sealingoperations were conducted in a bio-safety hood. The appropriate numberof vials for each formulation were placed in 2˜8° C., 25° C. and 40° C.stability chambers respectively. The formulations were frozen/thawed(−40±5° C./RT) for 5 cycles, and agitated at 300 rpm at 25° C. for 7days. Samples were drawn and analyzed at pre-determined time points.

The appearance, protein concentration, osmolality and pH value resultsof the freeze/thaw studies are summarized in Table 10. The proteinconcentration and osmolality were all around the target value at T0. ThepH value of the 9 samples were all around the target value after 5freeze/thaw cycles (−40±5° C./RT). The samples were all colorless,slightly opalescent and free of visible particles at T0. After 5freeze/thaw cycles (−40±5° C./RT), the opalescent level of F1 and F3samples were deeper than the other samples and a large number of visibleparticles were found in the F7 sample due to the absence of PS80. Thisdata suggested that anti-Cx43 Ab was relatively more stable in F2, F4,F5, F6, F8 and F9.

TABLE 10 Protein concentration, pH, osmolality and appearance resultsfrom the freeze/thaw study Protein concentration Osmolality mg/mLmOsm/kg pH Appearance No. T0 T0 T0 FT-5 C. T0 FT-5 C. F1 50.7 322 5.65.5 A A F2 51.6 311 5.5 5.4 A A F3 51.9 306 5.6 5.6 A A F4 50.3 331 5.65.5 A A F5 50.1 328 5.5 5.5 A A F6 50.0 342 5.5 5.5 A A F7 50.4 333 5.55.5 A C F8 25.8 311 5.4 5.4 A A F9 51.4 311 5.2 5.1 A A Notes: A =Colorless, slightly opalescent and free of visible particle; B =Colorless, slightly opalescent and slightly visible particles; C =Colorless, slightly opalescent and a large number of visible particles.

The MFI results of freeze/thaw are summarized in Table 11. The particlecounts in F7, with no PS80, was slightly more than the others at T0.After 5 freeze/thaw cycles (−40±5° C./RT), no growth trend was found forall formulations.

TABLE 11 MFI results from the freeze/thaw study MFI (Counts/mL)Formulation ECD ≥ 2 μm ECD ≥ 10 μm ECD ≥ 25 μm No. T0 FT-5C T0 FT-5 C.T0 FT-5 C. F1 3587 6575 68 92 9 2 F2 1452 2467 9 10 0 0 F3 1531 2602 1217 0 0 F4 2135 894 17 5 2 0 F5 576 1138 10 5 0 0 F6 1528 1577 14 14 0 2F7 6060 6028 97 40 4 2 F8 1954 2055 28 10 2 2 F9 458 1341 5 4 0 0

The SEC-HPLC results for all formulations are listed in Table 12 andFIG. 6. At T0, all formulations had similar SEC purity with the mainpeak around 99.5%. After 5 freeze/thaw cycles (−40±5° C./RT), allformulations had comparable SEC main peak purity greater than 99% exceptfor the F3 sample. The main peak purity decline in the F3 sample showeda slightly higher decrease at 2.7%.

TABLE 12 SEC-HPLC results from the freeze/thaw study SEC-HPLC resultsFormulation Main peak % HMW % LMW % No. T0 FT-5 C. T0 FT-5 C. T0 FT-5CF1 99.4 99.4 0.6 0.6 ND ND F2 99.4 99.5 0.6 0.6 ND ND F3 99.5 96.8 0.53.2 ND ND F4 99.5 99.5 0.5 0.6 ND ND F5 99.5 99.5 0.5 0.5 ND ND F6 99.599.5 0.6 0.5 ND ND F7 99.5 99.5 0.5 0.5 ND ND F8 99.5 99.5 0.5 0.5 ND NDF9 99.5 99.5 0.5 0.5 ND ND

The cIEF results for all formulations are listed in Table 13 and FIG. 7.There were no significant changes in pI. The main peak for samples F1,F7 and F9 was slightly lower than for the other formulations. Comparedto T0, the proportion of main peak, acidic peak and basic peak alsodisplayed no significant changes for all of the samples through 5freeze/thaw cycles (−40±5° C./RT).

TABLE 13 cIEF results from the freeze/thaw study cIEF results pI Mainpeak % Acidic peak % Basic peak % Formulation FT- FT- FT- FT- No. T0 5C. T0 5 C. T0 5 C. T0 5 C. F1 8.7 8.7 46.7 46.7 38.9 41.1 14.4 12.1 F28.7 8.7 47.9 47.0 38.7 40.8 13.4 12.2 F3 8.7 8.7 47.1 46.4 38.9 41.214.0 12.4 F4 8.7 8.7 48.2 47.1 38.5 40.7 13.3 12.2 F5 8.7 8.7 47.2 46.439.4 42.0 13.5 11.6 F6 8.7 8.7 48.1 46.4 38.4 40.8 13.6 12.8 F7 8.7 8.746.8 46.2 39.4 41.0 13.8 12.8 F8 8.7 8.7 47.3 46.9 39.4 41.1 13.3 12.0F9 8.7 8.7 46.6 46.2 40.2 41.1 13.3 12.7

The SDS-Caliper data for all formulations are listed in Table 14 andFIG. 8. All formulations showed stable purity in either non-reducedSDS-Caliper or reduced SDS-Caliper after 5 freeze/thaw cycles (−40±5°C./RT).

TABLE 14 SDS-Caliper results from the freeze/thaw study SDS-CaliperPurity Non-reduced SDS- Reduced SDS- Formulation Caliper Purity %Caliper Purity % No. T0 FT-5C T0 FT-5C F1 98.6 98.7 98.2 98.0 F2 98.698.8 98.1 98.1 F3 98.6 98.8 98.1 98.1 F4 98.6 98.7 98.2 98.2 F5 98.598.8 98.2 98.2 F6 98.5 98.7 98.1 98.1 F7 98.6 98.7 98.2 98.2 F8 98.698.7 98.1 98.1 F9 98.6 98.7 98.2 98.1

The appearance, protein concentration, osmolality and pH value resultsof the agitation study are summarized in Table 15. The proteinconcentration and osmolality were all around the target value at T0.Except for F7, all formulations remained stable in pH value andappearance after agitation at 25° C. for 7 days. The opalescent level ofthe F1 and F3 samples were deeper than the other samples and a largenumber of visible particles were found in the F7 sample after agitationat 25° C. for 7 days.

TABLE 15 Protein concentration, pH value, osmolality and appearanceresults from the agitation study Protein concentration Osmolality mg/mLmOsm/kg pH Appearance No. T0 T0 T0 A-7 D T0 A-7 D F1 50.7 322 5.6 5.5 AA F2 51.6 311 5.5 5.5 A A F3 51.9 306 5.6 5.5 A A F4 50.3 331 5.6 5.5 AA F5 50.1 328 5.5 5.5 A A F6 50.0 342 5.5 5.5 A A F7 50.6 333 5.5 5.5 AC F8 25.8 311 5.4 5.4 A A F9 51.4 311 5.2 5.1 A A Notes: A = Colorless,slightly opalescent and free of visible particle; B = Colorless,slightly opalescent and slightly visible particles; C = Colorless,slightly opalescent and a large number of visible particles.

The MFI data for all the samples is listed in Table 16. The particlecounts of F7 were slightly more than the others at T0. After 7-dayagitation at 25° C., there was slight growth trend of particle countsfor F7 due to the absence of PS80. Except for F7, all other samples hadsimilar particulate counts and no growth trend was found.

TABLE 16 MFI results from the agitation study MFI (Counts/mL) ECD ≥ 2 μmECD ≥ 10 μm ECD ≥ 25 μm Formulation A- A- A- No. T0 7 D T0 7 D T0 7 D F13587 2275 68 17 9 0 F2 1452 2219 9 63 0 0 F3 1531 4777 12 30 0 0 F4 21351895 17 15 2 0 F5 576 1652 10 7 0 0 F6 1528 1267 14 17 0 0 F7 6060 219897 156 4 30 F8 1954 1563 28 5 2 0 F9 458 484 5 4 0 0

The SEC-HPLC results for all formulations are listed in Table 17 andFIG. 9. After 7-day agitation at 25° C., all formulations had similarSEC main peak purity of more than 99%.

TABLE 17 SEC-HPLC results from the agitation study SEC-HPLC results Mainpeak % HMW % LMW % Formulation A- A- A- No. T0 7 D T0 7 D T0 7 D F1 99.499.3 0.6 0.7 ND ND F2 99.4 99.4 0.6 0.6 ND ND F3 99.5 99.5 0.5 0.5 ND NDF4 99.5 99.4 0.5 0.6 ND ND F5 99.5 99.5 0.5 0.6 ND ND F6 99.5 99.4 0.60.6 ND ND F7 99.5 99.1 0.5 0.9 ND ND F8 99.5 99.5 0.5 0.5 ND ND F9 99.599.5 0.5 0.5 ND ND

The cIEF results for all formulations are listed in Table 18 and FIG.10. There were no changes in pI for all samples after 7-day agitation at25° C. After 7-day agitation at 25° C., the main peak purity of allformulations remained stable except for F7. The main peak purity offormulation F7 declined 1.2%.

TABLE 18 cIEF results from the agitation study cIEF results Main peak %Acidic peak % Basic peak % Formulation pI value A- A- A- No. T0 A- T0 7D T0 7 D T0 7 D F1 8.7 8.7 46.7 46.1 38.9 42.1 14.4 11.9 F2 8.7 8.7 47.946.5 38.7 41.8 13.4 11.7 F3 8.7 8.7 47.1 46.7 38.9 41.7 14.0 11.6 F4 8.78.7 48.2 46.2 38.5 41.6 13.3 12.2 F5 8.7 8.7 47.2 46.2 39.4 41.9 13.511.9 F6 8.7 8.7 48.1 46.5 38.4 41.7 13.6 11.9 F7 8.7 8.7 46.8 45.6 39.441.7 13.8 12.7 F8 8.7 8.7 47.3 46.2 39.4 41.7 13.3 12.1 F9 8.7 8.7 46.646.7 40.2 41.2 13.3 12.1

The SDS-Caliper results for all formulations are listed in Table 19 andFIG. 11. All formulations showed stable purity in either non-reducedSDS-Caliper or reduced SDS-Caliper after 7-day agitation at 25° C.

TABLE 19 SDS-Caliper results from the agitation study SDS-Caliper PurityNon-reduced SDS- Reduced SDS- Formulation Caliper Purity % CaliperPurity % No. T0 A-7 D T0 A-7 D F1 98.6 98.7 98.2 98.0 F2 98.6 98.8 98.198.1 F3 98.6 98.6 98.1 97.9 F4 98.6 98.6 98.2 98.0 F5 98.5 98.6 98.298.0 F6 98.5 98.6 98.1 97.9 F7 98.6 98.7 98.2 98.0 F8 98.6 98.4 98.197.9 F9 98.6 98.5 98.2 98.0

The appearance, protein concentration, osmolality and pH value resultsfor the accelerated stability study are summarized in Table 20 and Table21. The protein concentration and osmolality were all around the targetvalue at T0. After storage at 2˜8° C., 25±2° C. and 40±2° C. for 4weeks, the pH values remained unchanged, while slightly visibleparticles were found in F7 due to the absence of PS80.

TABLE 20 Appearance results from the accelerated stability studyAppearance Formulation 05- 25- 25- 40- 40- No. T0 4 W 2 W 4 W 2 W 4 W F1A A A A A A F2 A A A A A A F3 A A A A A A F4 A A A A A A F5 A A A A A AF6 A A A A A A F7 A B B B B B F8 A A A A A A F9 A A A A A A Notes: A =Colorless, slightly opalescent and free of visible particle; B =Colorless, slightly opalescent and slightly visible particles; C =Colorless, slightly opalescent and a large number of visible particles.

TABLE 21 Protein concentration, osmolality and pH results from theaccelerated stability study Conc. Osmolality pH (mg/mL) (mOsm/kg) 05-25- 25- 40- 40- No. T0 T0 T0 4 W 2 W 4 W 2 W 4 W F1 50.7 322 5.6 5.6 5.55.6 5.6 5.6 F2 51.6 311 5.5 5.6 5.5 5.6 5.6 5.5 F3 51.9 306 5.6 5.6 5.65.6 5.6 5.6 F4 50.3 331 5.6 5.5 5.5 5.6 5.5 5.5 F5 50.1 328 5.5 5.6 5.55.6 5.6 5.5 F6 50.0 342 5.5 5.6 5.6 5.6 5.5 5.5 F7 50.6 333 5.5 5.6 5.65.6 5.6 5.6 F8 25.8 311 5.4 5.5 5.5 5.5 5.5 5.5 F9 51.4 311 5.2 5.2 5.25.2 5.2 5.2

The MFI data for all the samples is listed in Table 22. After storage at2˜8° C. and 25±2° C. for 4 weeks, there was no obvious changes insub-visible particles counts in all formulations. After storage at 40±2°C. for 4 weeks, the increases of sub-visible particle counts (ECD ≥10 μmand ECD ≥25 μm) in F7 was much higher than that in the otherformulations and there was a slight growth trend for particle counts inF7.

TABLE 22 MFI results from the accelerated stability study MFI(Counts/mL) ECD ≥ 2 μm ECD ≥ 10 μm ECD ≥ 25 μm 05- 25- 25- 40- 40- 05-25- 25- 40- 40- 05- 25- 25- 40- 40- No. T0 4 W 2 W 4 W 2 W 4 W T0 4 W 2W 4W 2W 4W T0 4W 2W 4W 2W 4W F1 3587 938 1806 324 1025 4038 68 9 12 2010 45 9 0 0 4 2 2 F2 1452 792 2101 2405 435 4287 9 7 10 20 7 25 0 0 4 40 0 F3 1531 579 1029 1437 654 1881 12 17 5 23 7 23 0 4 0 4 4 0 F4 2135913 550 1319 407 1351 17 19 2 15 7 25 2 0 0 0 4 7 F5 576 405 240 600 261761 10 9 4 7 2 12 0 0 2 2 0 4 F6 1528 655 404 1174 694 1414 14 12 14 1030 20 0 2 4 0 0 2 F7 6060 1566 2994 2700 2635 5707 97 33 74 50 199 425 42 4 2 12 53 F8 1954 457 492 943 956 1260 28 5 10 17 7 9 2 0 4 2 0 0 F9458 231 273 1467 240 921 5 4 5 23 4 12 0 0 4 0 0 2

The SEC-HPLC data for all the samples are listed in Table 23 and FIG.12. After storage at 2˜8° C. or 25±2° C. for 4 weeks, there was noobvious change in main peak purity in all formulations. After storage at40±2° C. for 2 weeks, significant decrease of the main peak wasobserved. After storage at 40±2° C. for 4 weeks, decline of the mainpeak was in the range of 2.2%˜4.9%. The decline of main peak purity inthe F1, F3 and F6 samples was 4.9%, 4.1% and 4.6% respectively. Incontrast, the main peak decline for F5 and F8 were relatively milderthan that for other formulations.

TABLE 23 SEC-HPLC results from the accelerated stability study SEC-HPLCresults 05- 25- 25- 40- 40- Formulation No. T0 4 W 2 W 4 W 2 W 4 W MainF1 99.4 99.3 99.2 99.1 96.6 94.5 peak % F2 99.4 99.4 99.3 99.2 97.3 96.5F3 99.5 99.5 99.3 99.2 96.3 95.4 F4 99.5 99.5 99.3 99.2 97.2 96.0 F599.5 99.5 99.3 99.3 97.5 97.2 F6 99.5 99.4 99.2 99.2 96.6 94.9 F7 99.599.5 99.3 99.3 97.5 97.0 F8 99.5 99.6 99.4 99.4 97.7 97.3 F9 99.5 99.599.3 99.3 97.4 97.0 HMW % F1 0.6 0.7 0.7 0.8 1.4 2.8 F2 0.6 0.6 0.7 0.70.9 1.2 F3 0.5 0.5 0.6 0.7 1.3 1.7 F4 0.5 0.6 0.6 0.7 0.9 1.4 F5 0.5 0.50.6 0.6 0.7 0.8 F6 0.6 0.6 0.7 0.7 1.1 2.1 F7 0.5 0.5 0.6 0.6 0.7 0.8 F80.5 0.4 0.5 0.5 0.6 0.6 F9 0.5 0.5 0.6 0.6 0.7 0.7 LMW % F1 ND ND 0.10.1 2.0 2.7 F2 ND ND 0.1 0.1 1.8 2.3 F3 ND ND 0.1 0.1 2.5 2.9 F4 ND ND0.1 0.1 1.9 2.6 F5 ND ND 0.1 0.1 1.8 2.1 F6 ND ND 0.1 0.1 2.3 3.0 F7 NDND 0.1 0.1 1.8 2.2 F8 ND ND 0.1 0.1 1.7 2.1 F9 ND ND 0.1 0.2 1.9 2.3

The cIEF data for all samples is listed in Table 24 and FIG. 13. Therewere no changes in pI for all samples stored at 2˜8° C., 25±2° C. or40±2° C. After storage at 2˜8° C. or 25±2° C. for 4 weeks, the main peakpurity for all samples declined slightly and there was no significantchanges in the main peak purity for all formulations. After storage at40±2° C. for 4 weeks, the main peak percentage for all samples declinedsignificantly, together with significantly increased acidic peak. Therewas no significant difference in main peak percentage for all thesamples, and the decline of the main peak was in the range of20.6%˜23.9%.

TABLE 24 cIEF results from the accelerated stability study cIEF results05- 25- 25- 40- 40- Formulation No. T0 4 W 2 W 4 W 2 W 4 W pI valueF1-F9 8.7 8.7 8.7 8.7 8.7 8.7 Main F1 46.7 44.6 45.3 43.5 36.0 25.2 peak% F2 47.9 45.5 46.2 43.6 35.2 25.8 F3 47.1 45.9 45.8 43.5 34.6 25.0 F448.2 45.5 46.8 44.0 35.8 24.9 F5 47.2 45.5 45.9 43.8 37.2 25.9 F6 48.145.1 46.8 43.7 34.1 24.2 F7 46.8 46.8 46.5 44.3 36.1 26.2 F8 47.3 46.346.3 44.1 37.6 26.0 F9 46.6 45.7 46.3 43.7 37.5 26.0 Acidic F1 38.9 42.240.8 42.8 49.7 63.3 peak % F2 38.7 42.2 41.0 43.3 51.9 63.0 F3 38.9 41.140.5 43.5 53.2 65.3 F4 38.5 41.5 39.5 42.6 51.3 63.7 F5 39.4 41.3 40.143.0 50.3 62.1 F6 38.4 41.6 39.1 43.2 52.4 65.2 F7 39.4 39.9 40.0 42.350.0 61.7 F8 39.4 40.5 40.0 42.4 49.2 61.7 F9 40.2 41.2 39.9 42.6 49.061.5 Basic F1 14.4 13.2 13.9 13.8 14.3 11.5 peak % F2 13.4 12.3 12.813.1 12.9 11.1 F3 14.0 12.9 13.7 13.0 12.3 9.7 F4 13.3 13.0 13.7 13.412.9 11.4 F5 13.5 13.2 14.0 13.2 12.5 12.0 F6 13.6 13.4 14.1 13.1 13.510.6 F7 13.8 13.4 13.5 13.4 13.8 12.1 F8 13.3 13.2 13.7 13.5 13.2 12.4F9 13.3 13.1 13.9 13.8 13.5 12.5

The SDS-Caliper data for all samples is listed in Table 25, FIG. 14 andFIG. 15. After storage at 2˜8° C. or 25±2° C. for 4 weeks, allformulations showed stable purity in non-reduced SDS-Caliper and reducedSDS-Caliper. After storage at 40±2° C. for 4 weeks, the purity of allformulations declined significantly in non-reduced SDS-Caliper andreduced SDS-Caliper. The non-reduced purity decline in F3 was 23.2%,which was the greatest reduction in all formulations. The purity declinein F1 and F3 was 6.0% and 6.1% respectively. The purity of F5 and F9 wasslightly higher than others according to the non-reduced SDS-Caliperdata, while the purity of F2 and F9 was slightly higher than othersaccording to the reduced SDS-Caliper data.

TABLE 25 SDS-Caliper results from the accelerated stability studySDS-Caliper Purity 05- 25- 25- 40- 40- Formulation No. T0 4 W 2 W 4 W 2W 4 W Non-reduced F1 98.6 98.4 98.2 97.8 87.7 77.8 SDS-Caliper F2 98.698.5 98.2 97.9 87.1 76.5 Purity % F3 98.6 98.4 98.2 97.6 85.0 75.4 F498.6 98.4 98.3 97.8 86.5 76.8 F5 98.5 98.4 98.3 97.8 86.0 78.2 F6 98.598.3 98.2 97.8 85.4 77.9 F7 98.6 98.4 98.2 97.7 84.9 76.7 F8 98.6 98.498.3 98.1 87.8 77.2 F9 98.6 98.4 98.3 98.1 87.9 79.4 Reduced F1 98.298.0 97.7 97.5 95.8 92.2 SDS-Caliper F2 98.1 98.2 97.7 97.7 95.7 93.8Purity % F3 98.1 98.2 97.6 97.6 95.4 92.0 F4 98.2 98.3 97.7 97.6 95.992.7 F5 98.2 98.2 97.7 97.6 95.7 92.9 F6 98.1 98.1 97.7 97.5 95.7 92.8F7 98.2 98.1 97.7 97.9 95.7 92.9 F8 98.1 98.2 97.8 97.5 96.6 93.0 F998.2 98.2 97.9 97.8 96.6 94.2

After 5 freeze/thaw cycles (−40±5° C./RT), anti-Cx43 Ab in allformulations had no significant difference in protein concentration, pHvalue, osmolality and purity (SDS-Caliper Reduced & Non-Reduced). Theopalescent level of the F 1 and F3 samples was deeper than the othersamples. Visible particles and sub-visible particle counts (MFI) insample F7 without surfactant were much greater than in the otherformulations, but no growth trend was found after 5 freeze/thaw cycles.The SEC main peak of the F3 sample showed a marginally higher decreaseat 2.7%. The cIEF main peak changed little among all formulations.

After 7-day agitation at 25° C., anti-Cx43 Ab in all formulations had nosignificant difference in protein concentration, pH value, osmolalityand purity (SEC-HPLC, cIEF, SDS-Caliper Reduced & Non-Reduced). Inaddition, the opalescent level of the F1 and F3 samples was deeper thanthe other samples. Visible particles and sub-visible particle counts(MFI) in sample F7 without surfactant were much greater than otherformulations.

After storage at 2˜8° C. for 4 weeks, anti-Cx43 Ab in all formulationshad no significant difference in protein concentration, pH value,osmolality, sub-visible particles and purity (SEC-HPLC, cIEF,SDS-Caliper Reduced & Non-Reduced). Only slightly visible particles werefound in formulation F7 after storage at 2˜8° C. for 4 weeks.

After storage at 25±2° C. for 4 weeks, anti-Cx43 Ab in all formulationshad no significant difference in protein concentration, pH value,osmolality, sub-visible particles and purity (SEC-HPLC, SDS-CaliperReduced & Non-Reduced). In addition, slightly visible particles werefound in formulation F7 because there was no addition of PS80. The cIEFmain peak of all samples declined significantly, but no significantdifference was found in nine formulations after storage at 25±2° C. for4 weeks.

After storage at 40±2° C. for 4 weeks, anti-Cx43 Ab in all formulationshad no significant difference in protein concentration, pH value andosmolality. Slightly visible particles were found in formulation F7because there was no addition of PS80. The increase of sub-visibleparticle counts (ECD ≥10 μm and ECD ≥25 μm) in F7 was much higher thanthat in the other formulations. The purity (SEC-HPLC, cIEF, SDS-CaliperReduced & Non-Reduced) of all the samples declined significantly. TheSEC main peak for F1, F3 and F6 declined significantly more than for theother formulations. The cIEF main peak of all the samples showed nosignificant difference. The non-reduced purity decline in F3 was thegreatest in all formulations. The purity decline in F1 and F3 was higherthan others.

In summary, formulation development studies including pH/Bufferscreening, excipients and PS80 strength screening were performed todetermine the lead formulation.

In pH/buffer screening, the histidine/histidine hydrochloride buffersystem exhibited better capability for stabilizing the protein.

In the excipients and PS80 strength screening, sodium chloride,sorbitol, glycine and sucrose (F1, F2, F3 and F4) were chosen toinvestigate the stability of anti-Cx43 Ab. The results suggested thatthe anti-Cx43 Ab was relatively more stable in histidine buffer withsucrose as excipient. The stability data of samples with differentconcentrations of PS80 (F4, F6 and F7) showed that 0.02% PS80 stabilizedthe anti-Cx43 Ab better than 0% or 0.05% PS80.

The SDS-Caliper data from the pH/buffer screening (Table 7) showed thatthe anti-Cx43 Ab was truncated after storage at 40±2° C. for 2 or 4weeks, which was confirmed by the generation of a LMW peak in theSEC-HPLC data. However, the data from the excipient and PS80 strengthscreening indicated that the addition of excipients did not alleviatethe protein truncation. Analysis by peptide mapping showed that theprotein was cleaved at the Asn-Pro site of the complementaritydetermining region (CDR) of the heavy chain. Furthermore, severalstudies have shown that the breakage of Asn-Pro is a hydrolysis reactionwhich happens due to the existence of water, and is accelerated underbasic environment or higher temperature conditions. The SDS-Caliper datafrom pH/buffer screening showed that the lowest purity sample was B9which was also the highest pH value (Table 7). Although the pH/buffersystem chosen for excipient screening was acidic, protein truncationcould not be prevented. The solution to minimize the truncation was todevelop the Ab in frozen DP form.

The protein concentration study containing F5 (50 mg/mL) and F8 (25mg/mL) showed that concentration had little effect on the stability ofthe anti-Cx43 Ab. Although the performance of F5 (with EDTA) wasslightly better than F4 (without EDTA) via SEC-HPLC, Caliper-SDS andcIEF tests at 40±2° C., the differences between these two formulationswas negligible under low temperature conditions at 25±2° C. and 2˜8° C.

In summary, 50 mg/mL of the anti-Cx43 Ab in 20 mM histidine/histidinehydrochloride at pH 5.5 with 8% sucrose and 0.02% (w/v) PS80 wasconsidered the lead formulation for the confirmation study.

Example 4: Formulation Confirmation Study

The anti-Cx43 Ab formulation confirmation study was performed to confirmthe stability of the selected formulation. The conditions evaluated inthe confirmation study include storage conditions, stress conditions,freeze/thaw and agitation. The formulation selected from the formulationscreening study was 50 mg/mL anti-Cx43 Ab in 20 mM histidine/histidinehydrochloride buffer at pH 5.5 with 8% (w/v) sucrose and 0.02% (w/v)PS80.

Stability studies were conducted for the selected lead formulation,which was 20 mM histidine/histidine hydrochloride buffer, 8% (w/v)sucrose, 0.02% (w/v) PS80 at pH 5.5, generated from first 15L pool (Lot:2144SD181129K01X01D01). The anti-Cx43 Ab concentration in this study was50 mg/mL. The formulations were stored at −40° C., −20° C., 2˜8° C. and25° C. for 3 months and stored at 40° C. for 4 weeks, respectively.Formulations were frozen/thawed (−40±5° C./RT) for 5 cycles, andagitated at 100 rpm at 25° C. for 7 days. Samples were pulled timely ateach time point and kept at 2˜8° C. before analysis. Testing itemsincluding appearance, pH, osmolality, Conc-UV280, SEC-HPLC, CEX-HPLC,CE-SDS (R&NR), MFI, mDSC and potency were performed for this study. Thesampling plan is shown in Table 26.

TABLE 26 Study Parameters from the anti-Cx43 Ab Formulation ConfirmationStudy DP (2144 150 mg/3 mL/vial; 6R glass vial) −40° Agitation −40° C.−20° C. 2-8° C. 25° C. 40° C. C.~RT 100 rpm T0 1 M 3 M 1 M 3 M 1 M 3 M 1M 2 M 3 M 2 wks 4 wks FT-5 C. 25-A-7 D x, y, z x x, z x x, z x x, z x xx, z x x, z x x Notes: x = Appearance, pH, Osmolality, Conc-UV280,SEC-HPLC, CEX-HPLC, CE-SDS (R&NR), MFI; y = mDSC; z = Potency.

Anti-Cx43 Ab DS was filtered through a 0.22 μm filter (Millipore ExpressPVDF Membrane) and then distributed into 6R vials with 3 mL/vial fillingvolume. Vials were immediately stoppered and sealed after filling. Allthe filtration, filling and sealing operations were conducted in abio-safety hood. The appropriate number of vials were placed in −40° C.,−20° C. and 2˜8° C. refrigerators, and placed in 25° C. and 40° C.stability chambers, respectively. Meanwhile, bottles were frozen in a−40° C. freezer and thawed at room temperature for 5 cycles and fixed to100 rpm thermostat shaker at 25° C. for 7 days, respectively. Sampleswere pulled and analyzed at pre-determined time points.

The thermogram of the anti-Cx43 Ab in 20 mM histidine/histidinehydrochloride at pH 5.5 with 8% sucrose and 0.02% (w/v) PS80 is shown inTable 27 and FIG. 16. The Tg′ onset value, the temperature at which thesample starts to glass transition, was considered an indicator for thestorage condition for the sample. The Tg′ onset of the anti-Cx43 Ab was−29.22° C.

TABLE 27 mDSC data from the anti-Cx43 Ab formulation confirmation studyTg′ onset (° C.) Tg′ middle (° C.) Tg′ end (° C.) −29.22 −28.60 −28.00

The appearance, protein concentration, pH and osmolality results of thefreeze/thaw and agitation studies are summarized in Table 28. There wasno obvious changes in the appearance, protein concentration, pH andosmolality after 5 freeze/thaw cycles (−40±5° C./RT) and 7-dayagitation. All samples appeared colorless, slightly opalescent and freeof visible particles. No obvious change was observed in proteinconcentration, and all results were within the specification of 50.0±5.0mg/mL. No obvious changes were observed in pH and osmolality compared toT0. The lower osmolality was caused by insufficient addition of sucroseduring DS preparation. The final concentration of sucrose in theconfirmation study was 6.7%. Theoretically, protein formulated with 8.0%sucrose would be more stable than protein formulated with 6.7% sucrose.It has been reported that Tm increases along with the escalation ofsucrose concentration. The stabilization effect of sucrose is aconsequence of the high cohesive force of the solvent, due to the stronginteraction between water and sucrose, excluding the protein from thesystem and leading to the stabilization of the folded protein. Thus, theincrease of Tm with sucrose concentration reflects the need for higherenergy to form a cavity in the water-sucrose mixture to accommodate theprotein molecule.

TABLE 28 The appearance, protein concentration, pH and osmolalityresults from the freeze-thaw and agitation studies Item T0 FT-5 C. A-7 DAppearance A A A Concentration (mg/mL) 45.8 45.7 46.0 pH 5.6 5.6 5.6Osmolality (mOSm/kg) 274 272 273 Notes: A = Colorless, slightlyopalescent and free of visible particles.

The MFI results for the freeze/thaw and agitation studies are summarizedin Table 29. No growth trend of particle counts (ECD ≥10 μm and ECD ≥25μm) was observed after 5 freeze/thaw cycles (−40±5° C./RT) and 7-dayagitation at 25° C.

TABLE 29 MFI data from the freeze-thaw and agitation studiesConcentration (#/mL) ECD >= 2 μm ECD >= 10 μm ECD >= 25 μm FT- A- FT- A-FT- A- NO. T0 5 C. 7 D T0 5 C. 7 D T0 5 C. 7 D F4 97 2836 2863 12 37 144 0 0

The SEC-HPLC results of the freeze/thaw and agitation studies aresummarized in Table 30. No obvious change was observed after 5freeze/thaw cycles and 7-day agitation. All samples maintained SEC mainpeak purity at 99.6% or 99.7%.

TABLE 30 SEC data from the freeze-thaw and agitation studies Main Peak %HMW Peak % LMW Peak % FT- A- FT- A- FT- A- NO T0 5 C. 7 D T0 5 C. 7 D T05 C. 7 D F4 99.7 99.7 99.6 0.2 0.3 0.3 0.0 0.1 0.1

The CE-SDS results for the freeze/thaw and agitation studies aresummarized in Table 31. No obvious changes were observed after 5freeze/thaw cycles and 7-day agitation.

TABLE 31 CE-SDS data from the freeze-thaw and agitation studies CE-RPurity % CE-NR Purity % CE-NR Pre-peaks % FT- A- FT- A- FT- A- NO T0 5C. 7 D T0 5 C. 7 D T0 5 C. 7 D F4 98.7 98.3 98.5 97.7 97.6 97.6 0.9 0.70.8

The CEX-HPLC results from the freeze/thaw and agitation studies aresummarized in Table 32. Compared to T0, the proportion of main peak,acidic peak and basic peak had no significant changes through 5freeze/thaw cycles (−40±5° C./RT) and 7-day agitation.

TABLE 32 CEX data from the freeze-thaw and agitation studies Main Peak %Acid Peak % Basic Peak % FT- A- FT- A- FT- A- NO T0 5 C. 7 D T0 5 C. 7 DT0 5 C. 7 D F4 70.4 72.5 72.1 20.2 17.9 18.1 9.3 9.6 9.8

The appearance, protein concentration, pH and osmolality results ofdifferent storage conditions are summarized in Table 33. All the sampleswere colorless, slightly opalescent and free of visible particles,except one sample stored at 25±2° C. for 1 month appeared to containsome visible particles by accident. No obvious change was observed inprotein concentration, pH and osmolality compared to T0, and all resultswere within the specification.

The MFI results of different storage conditions are summarized in Table34. There was no obvious change in the sub-visible particle counts at−40° C., −20° C., 2˜8° C. and 25° C. The amount of sub-visible particlesincreased significantly after storage at 40±2° C. for 2 weeks, but nogrowth trend was found after storage at 40±2° C. for 4 weeks. The dataat 25±2° C. for 1 month was for reference due to the generation ofvisible particles.

The SEC-HPLC results of samples under different storage conditions aresummarized in Table 35. No obvious changes were observed after storageat −40±5° C., −20±5° C. and 2˜8° C. for 3 months. All samples maintainedSEC main peak purity at 99.6% or 99.7%. For samples incubated at 25° C.,a slight decline of the main peak (drop of main peak was equal to 2.0%)was observed after 3 months of storage. For samples incubated at 40° C.,a significant decrease of main peak (drop of main peak was equal to4.2%) was observed after 4 weeks of storage.

The CE-SDS results for different storage conditions are shown in Table36. No obvious changes were observed after storage at −40±5° C., −20±5°C. and 2˜8° C. for 3 months. All samples maintained CE-R and CE-NR mainpeak purity at the range of 98.6%˜99.3% and 97.0%˜98.1%, respectively.For samples incubated at 25° C., a slight decline of main peak wasobserved after 1 month of storage, and a significant decrease of mainpeak (drop of CE-R main peak was equal to 4.4% and drop of CE-NR mainpeak was equal to 10.5%) was observed after 3 months of storage. Forsamples incubated at 40° C., a significant decrease of main peak (dropof CE-R main peak was equal to 7.7% and drop of CE-NR main peak wasequal to 22.8%) was observed after 4 weeks of storage.

The CEX-HPLC results for different storage conditions are shown in Table37. No obvious changes were observed after storage at −40±5° C., −20±5°C. and 2˜8° C. for 3 months. All samples maintained CEX main peak at therange of 70.4%˜73.8%. For samples incubated at 25° C., a slight declineof main peak was observed after 1 month of storage, and a significantdecrease of main peak (drop of CEX main peak was equal to 10.9%) wasobserved after 3 months of storage. For samples incubated at 40° C., asignificant decrease of main peak (drop of CEX main peak was equal to25.9%) was observed after 4 weeks of storage.

The potency results for different storage conditions are shown in Table38. The potency for different storage conditions remained in the rangeof 60˜140%. No potency decline was observed for the formulations duringthe study after storage at −40±5° C., −20±5° C., 2˜8° C. and 25±2° C.for 3 months. A significant decline in potency was observed afterstorage at 40° C. for 4 weeks.

After 5 freeze/thaw cycles (−40±5° C./RT) and 7-day agitation at 25° C.,the anti-Cx43 Ab in the selected formulation had no significantdifference in appearance, protein concentration, pH value, osmolalityand purity (SEC-HPLC, CEX-HPLC, CE-SDS Reduced & Non-Reduced). Afterstorage at −40+5° C., −20+5° C. and 2˜8° C. for 3 months, the anti-Cx43Ab in the selected formulation had no significant difference inappearance, protein concentration, pH value, osmolality, purity(SEC-HPLC, CEX-HPLC, CE-SDS Reduced & Non-Reduced) and potency. Afterstorage at 25+2° C. for 3 months, the anti-Cx43 Ab in the selectedformulation had no significant difference in appearance, proteinconcentration, pH value, osmolality and potency. The purity (SEC-HPLC,CEX-HPLC, CE-SDS Reduced & Non-Reduced) of the selected formulationdeclined slightly after storage at 25±2° C. for 1 month and declinedsignificantly after storage at 25±2° C. for 3 months. After storage at40+2° C. for 4 weeks, the anti-Cx43 Ab in the selected formulation hadno significant difference in appearance, protein concentration, pH valueand osmolality. The amount of sub-visible particles increasedsignificantly after storage at 40±2° C. for 2 weeks, but no growth trendwas found after storage at 40±2° C. for 4 weeks. The purity (SEC-HPLC,CEX-HPLC, CE-SDS Reduced & Non-Reduced) and potency of the selectedformulation all declined significantly.

In summary, 50 mg/mL anti-Cx43 Ab in 20 mM histidine/histidinehydrochloride at pH 5.5 with 8% sucrose and 0.02% (w/v) PS80 wasselected as the lead formulation for the confirmation study. All itemstested displayed no significant change when −20+5° C. was the long-termstorage temperature, indicating that the anti-Cx43 Ab was stable in thisformulation.

TABLE 33 The appearance, protein concentration, pH and osmolalityresults from the different storage temperature study −40° C. −20° C. 5°C. 25° C. 40° C. Item T0 1 M 2 M 3 M 1 M 2 M 3 M 1 M 3 M 1 M 2 M 3 M 2 W4 W Appearance A A A A A A A A A B A A A A Concentration 45.8 45.0 47.345.9 45.1 48.7 46.0 45.4 46.1 45.4 45.9 45.3 45.2 45.4 (mg/mL) pH 5.65.5 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6 5.6 Osmolality 274 270265 262 265 268 260 264 265 268 279 269 268 272 (mOSm/kg) Notes: A -Colorless, slightly opalescent and free of visible particles; B =Colorless, slightly opalescent and visible particles.

TABLE 34 MFI data from the different storage temperature studyConcentration −40° C. −20° C. 5° C. 25° C. 40° C. (#/mL) T0 1 M 2 M 3 M1 M 2 M 3 M 1 M 3 M 1 M 2 M 3 M 2 W 4 W ECD> = 2 μm 97 2654 1994 25961792 1832 1170 1380 6917 16591 3887 3772 15633 6000 ECD> = 10 μm 12 5017 28 37 20 10 19 133 187 28 71 371 100 ECD> = 25 μm 4 5 2 4 0 0 0 0 212 5 9 19 4

TABLE 35 SEC data from the different storage temperature study −40° C.−20° C. 5° C. 25° C. 40° C. SEC (%) T0 1 M 2 M 3 M 1 M 2 M 3 M 1 M 3 M 1M 2 M 3 M 2 W 4 W Main peak 99.7 99.7 99.7 99.7 99.7 99.7 99.7 99.6 99.698.9 98.3 97.7 97.2 95.5 HMW 0.2 0.3 0.3 0.3 0.3 0.3 0.3 0.4 0.3 0.4 0.50.8 1.0 1.7 LMW 0.0 0.0 0.0 0.0 0.1 0.0 0.0 0.1 0.0 0.8 1.2 1.5 1.8 2.8

TABLE 36 CE data from the different storage temperature study −40° C.−20° C. 5° C. 25° C. 40° C. CE % T0 1 M 2 M 3 M 1 M 2 M 3 M 1 M 3 M 1 M2 M 3 M 2 W 4 W CE-R purity 99.3 99.0 98.7 98.8 98.8 98.8 98.9 98.6 98.798.4 96.2 94.9 94.7 91.6 CE-NR 97.7 97.4 97.7 98.1 97.0 97.7 98.0 97.598.1 92.8 89.9 87.2 87.8 74.9 purity CE-NR 0.9 1.1 0.7 0.7 1.2 0.7 0.70.7 0.8 5.6 8.5 11.0 11.6 24.2 pre-peak

TABLE 37 CEX data from the different storage temperature study −40° C.−20° C. 5° C. 25° C. 40° C. CEX % T0 1 M 2 M 3 M 1 M 2 M 3 M 1 M 3 M 1 M2 M 3 M 2 W 4 W Main peak 70.4 72.5 72.6 73.5 72.4 72.6 73.8 72.3 73.767.9 62.9 59.5 53.7 44.5 Acidic 20.2 18.0 18.1 17.4 18.1 18.0 17.3 18.217.5 21.1 26.6 30.2 34.0 43.5 peak Basic peak 9.3 9.5 9.3 9.0 9.5 9.39.0 9.5 8.8 11.0 10.5 10.3 12.3 11.9

TABLE 38 Potency data from the different storage temperature studyPotency −40° C. −20° C. 5° C. 25° C. 40° C. NO. T0 3 M 3 M 3 M 1 M 3 M 2W 4 W Confir- 97% 104% 116% 93% 90% 82% 93% 62% mation

Modifications

Modifications and variations of the described methods and compositionsof the present disclosure will be apparent to those skilled in the artwithout departing from the scope and spirit of the disclosure. Althoughthe disclosure has been described in connection with specificembodiments, it should be understood that the disclosure as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out thedisclosure are intended and understood by those skilled in the relevantfield in which this disclosure resides to be within the scope of thedisclosure as represented by the following claims.

INCORPORATION BY REFERENCE

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A pharmaceutical formulation comprising: an anti-Cx43 antibody orantigen binding fragment thereof; a buffer; a surfactant; and astabilizer; wherein the pharmaceutical formulation has a pH of betweenabout 5 and about 6; wherein the anti-Cx43 antibody or antigen bindingfragment thereof comprises: a first, second and third heavy chaincomplementarity determining region (CDR) sequence having the amino acidsequence of SEQ ID NOs: 1, 2, and 3, respectively; and a first, secondand third light chain CDR sequence having the amino acid sequence of SEQID NOs: 4, 5, and 6, respectively.
 2. The pharmaceutical formulation ofclaim 1, wherein the anti-Cx43 antibody or antigen binding fragmentthereof comprises a heavy chain variable domain having the amino acidsequence of SEQ ID NO: 7, and a light chain variable domain having theamino acid sequence of SEQ ID NO:
 8. 3. The pharmaceutical formulationof claim 2, wherein the anti-Cx43 antibody or antigen binding fragmentthereof comprises a heavy chain having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 9-17, and a light chain havingthe amino acid sequence of SEQ ID NO:
 18. 4. The pharmaceuticalformulation of claim 1, wherein the anti-Cx43 antibody or antigenbinding fragment thereof binds to an epitope located within the aminoacid sequence of FLSRPTEKTI (SEQ ID NO: 19).
 5. The pharmaceuticalformulation of claim 4, wherein the epitope comprises one or more aminoacids selected from the group consisting of R4, P5, E7, K8 and I10 ofSEQ ID NO:
 19. 6. The pharmaceutical formulation of claim 4, wherein theepitope consists of R4, P5, E7, K8 and I10 of SEQ ID NO:
 19. 7. Thepharmaceutical formulation of claim 4, wherein the epitope comprises allten amino acids of SEQ ID NO:
 19. 8. The pharmaceutical formulation ofclaim 4, wherein the epitope consists of all ten amino acids of SEQ IDNO:
 19. 9. The pharmaceutical formulation of claim 1, wherein theanti-Cx43 antibody or antigen binding fragment thereof is present at aconcentration of between about 5 and about 100 mg/mL, preferably between20 and 80, more preferably 40 to 60 mg/mL.
 10. The pharmaceuticalformulation of claim 1, wherein the buffer is selected fromacetate/sodium acetate, histidine/aspartic acid, citric acid/sodiumcitrate, dibasic sodium phosphate/sodium dihydrogen phosphate, andhistidine/histidine hydrochloride.
 11. The pharmaceutical formulation ofclaim 10, wherein the buffer is histidine/aspartic acid orhistidine/histidine hydrochloride.
 12. The pharmaceutical formulation ofclaim 11, wherein the buffer is histidine/histidine hydrochloride. 13.The pharmaceutical formulation of claim 1, wherein the surfactant ispolysorbate 80 (PS80).
 14. The pharmaceutical formulation of claim 1,wherein the stabilizer is selected from ethylenediaminetetraacetic acid(EDTA), sodium chloride, sorbitol, glycine, and sucrose.
 15. Thepharmaceutical formulation of claim 14, wherein the stabilizer issucrose.
 16. The pharmaceutical formulation of claim 1, wherein the pHis between about 5.4 to about 5.6.
 17. The pharmaceutical formulation ofclaim 1, wherein the formulation is an aqueous formulation.
 18. Apharmaceutical formulation comprising: about 40-60 mg/mL, preferablyabout 50 mg/mL of an anti-Cx43 antibody or antigen binding fragmentthereof; about 10-40 mM, preferably about 20 mM histidine/histidinehydrochloride buffer; about 0.005%-0.05%, preferably about 0.02% w/vPolysorbate 80; and about 1%-20% w/v, preferably about 8% w/v sucrose;wherein the formulation has a pH of between about 5.4 to about 5.6,preferably about 5.5.
 19. A pharmaceutical formulation comprising: about50 mg/mL an anti-Cx43 antibody or antigen binding fragment thereof,comprising a heavy chain having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 9-17, and comprising a light chainhaving the amino acid sequence of SEQ ID NO: 18; about 20 mMhistidine/aspartic acid buffer; about 0.02% w/v Polysorbate 80; andabout 8% w/v sucrose, wherein the formulation has a pH of between about5.4 to about 5.6, preferably about 5.5.
 20. A method of inhibitingopening of Cx43 hemichannels in cells, comprising administering to asubject in need thereof the pharmaceutical formulation of claim 1,preferably for treating an inflammatory disease or condition or aneurodegenerative disease such as spinal cord injury.